Conjugated anti-cd38 antibodies

ABSTRACT

Isolated antibodies that bind to human CD38 and cynomolgus CD38 are disclosed. Also disclosed are pharmaceutical compositions comprising the disclosed antibodies, and therapeutic and diagnostic methods for using the disclosed antibodies.

This application is a continuation of U.S. patent application Ser. No.15/598,241, filed May 17, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/754,592, filed Jun. 29, 2015, now U.S. Pat. No.9,676,869, which is a continuation of U.S. patent application Ser. No.13/977,207, filed Feb. 13, 2014, now U.S. Pat. No. 9,102,744, which is a371 National Phase application of PCT/US11/68244 filed Dec. 30, 2011,which claims benefit under 35 U.S.C. § 119(e) to U.S. Ser. No.61/428,699, filed Dec. 30, 2010; U.S. Ser. No. 61/470,382, filed Mar.31, 2011; U.S. Ser. No. 61/470,406, filed Mar. 31, 2011; and U.S. Ser.No. 61/485,104, filed May 11, 2011, all entirely incorporated byreference.

BACKGROUND

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 14, 2019, isnamed 101588-5008-US03-Sequence-Listing.txt and is 55,959 bytes in size.

CD38, also known as cyclic ADP ribose hydrolase, is a type IItransmembrane glycoprotein with a long C-terminal extracellular domainand a short N-terminal cytoplasmic domain. CD38 is a member of a groupof related membrane bound or soluble enzymes, comprising CD157 andAplysia ADPR cyclase. This family of enzymes has the unique capacity toconvert NAD to cyclic ADP ribose or nictotinic acid-adenine dinucleotidephosphate.

In addition, CD38 has been reported to be involved in Ca²⁺ mobilizationand in the signal transduction through tyrosine phosphorylation ofnumerous signaling molecules, including phospholipase Cγ, ZAP-70, syk,and c-cbl. Based on these observations, CD38 was proposed to be animportant signaling molecule in the maturation and activation oflymphoid cells during their normal development.

Among hematopoietic cells, an assortment of functional effects have beenascribed to CD38 mediated signalling, including lymphocyteproliferation, cytokine release, regulation of B and myeloid celldevelopment and survival, and induction of dendritic cell maturation.

Yet, the exact role of CD38 in signal transduction and hematopoiesisremains unclear, since most of the signal transduction studies have usedcell lines ectopically overexpressing CD38 and anti-CD38 monoclonalantibodies, which are non-physiological ligands.

The presumed natural ligand of CD38 is CD31 (PECAM-1; PlateletEndothelial Cell Adhesion Molecule-1). CD31 is a 130 kD member of theimmunoglobulin superfamily which is expressed on the surface ofcirculating platelets, neutrophils, monocytes, and naïve B-lymphocytes.Functionally, CD31 is thought to act as an adhesion molecule. It hasbeen suggested that the interaction of CD38 with CD31 may act inpromoting survival of leukemia cells.

Animal models deficient for a single molecule have in many instancesbeen fundamental tools for understanding the biological role of themolecule in the animal. The underlying assumption is that if the proteinexerts a non-redundant function, then its complete lack will result inthe complete loss of that function.

CD38 knockout mice have been generated. These animals show an almostcomplete loss of tissue associated NADase activity. Yet, these animalsare viable, leading to the conclusion that CD38 and its activities arenot necessary for life. These mice do however exhibit a defect in theirinnate immunity and a reduced T-cell dependent humoral response.

In contrast to the results in mice, in humans there is strongcircumstantial evidence that the absence of CD38 is incompatible withlife. Analysis of more than 5,000 blood samples from newborns failed toidentify a single CD38⁻ individual; suggesting that unlike mice, CD38 isnecessary for survival. Thus, it is not clear that the observations madein mice concerning CD38 function can be extrapolated to humans.

CD38 is upregulated in many hematopoeitic malignancies and in cell linesderived from various hematopoietic malignancies including non-Hodgkin'slymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), Bchronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia(ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cellleukemia (HCL), Hodgkin's Lymphoma (HL), and chronic myeloid leukemia(CML). On the other hand, most primitive pluripotent stem cells of thehematopoietic system are CD38⁻ (FIG. 1).

In spite of the recent progress in the discovery and development ofanti-cancer agents, many forms of cancer involving CD38-expressingtumors still have a poor prognosis. Thus, there is a need for improvedmethods for treating such forms of cancer.

BRIEF SUMMARY OF THE INVENTION

Provided herein are reagents and methods for binding to CD38 andmethods, for treating CD38 associated diseases and detecting CD38 usingCD38-specific binding agents including antibodies specific for CD38. Fortherapeutic purposes, the antibodies of the invention include aconjugated drug moiety, as described below. For diagnostic purposes, theantibodies of the invention can optionally include a detectable label.

Accordingly, in some embodiments, an isolated antibody specific forhuman CD38 (SEQ ID NO:1) and cynomolgus CD38 (SEQ ID NO:2) is described.This antibody is composed of a heavy chain variable region and a lightchain variable region, wherein the heavy chain variable region iscomposed of three complementary determining regions (CDRs), HCDR1,HCDR2, and HCDR3, and wherein the light chain variable region is alsocomposed of three CDRs, LCDR1, LCDR2, and LCDR3. The sequences of theCDRs are represented by: HCDR1 (SEQ ID NO:3), HCDR2 (SEQ ID NO:4), HCDR3(SEQ ID NO:5), LCDR1 (SEQ ID NO:6), LCDR2 (SEQ ID NO:7) and LCDR3 (SEQID NO:8). In some embodiments, the antibody further comprises aconjugated drug moiety.

In other embodiments, the isolated antibody is composed of a heavy chainvariable region, wherein the sequence of heavy chain variable region isencompassed by SEQ ID NO:9. In other embodiments, the isolated antibodyis composed of a light chain variable region, wherein the sequence ofthe light chain variable region is encompassed by SEQ ID NO:10. In someembodiments, the antibody further comprises a conjugated drug moiety.

In some embodiments, the isolated antibody is composed of a heavy chainvariable region, wherein the sequence of heavy chain variable region isencompassed by SEQ ID NO:9. In other embodiments, the isolated antibodyis composed of a light chain variable region, wherein the sequence ofthe light chain variable region is encompassed by SEQ ID NO:10. Thiscombination of heavy chain variable region and light chain variableregion is referred to as Ab79. In some embodiments, the antibody furthercomprises a conjugated drug moiety.

In some embodiments, the isolated antibody is composed of a heavy chainand a light chain, wherein the heavy chain sequence is encompassed bySEQ ID NO:11 and the light chain is encompassed by SEQ ID NO:12. In someembodiments, the antibody further comprises a conjugated drug moiety.

In some embodiments, the isolated antibody includes an Fc domain. Inother embodiments, the Fc domain is a human Fc domain. In still otherembodiments, the Fc domain is a variant Fc domain.

In some embodiments, an isolated nucleic acid encoding the heavy chainof SEQ ID NO:11 is provided. In other embodiments, an isolated nucleicacid encoding the light chain of SEQ ID NO:12 is provided.

In some embodiments, a host cell is provided, the host cell containingthe isolated nucleic acid encoding the heavy chain of SEQ ID NO:11 andthe isolated nucleic acid encoding the light chain of SEQ ID NO:12.

In some embodiments, a method of producing the antibody of the inventionis provided. The method encompassing culturing a host cell containingthe isolated nucleic acid encoding the heavy chain of SEQ ID NO:11 andthe isolated nucleic acid encoding the light chain of SEQ ID NO:12 underconditions wherein the isolated nucleic acid(s) are expressed and anantibody is produced. The drug moiety is then attached to the antibodyusing chemistries standard in the art.

In some embodiments, an isolated antibody specific for human CD38 (SEQID NO:1) and cynomolgus CD38 (SEQ ID NO:2) is described. This antibodyis composed of six CDRs, wherein each CDR of this antibody can differfrom SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,and SEQ ID NO:8 by 0, 1, or 2 amino acid substitutions. In someembodiments, the antibody further comprises a conjugated drug moiety.

In other embodiments, an isolated antibody specific for human CD38 (SEQID NO:1) and cynomolgus CD38 (SEQ ID NO:2) is described. This antibodyis composed of a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region is composed of threecomplementary determining regions (CDRs), HCDR1, HCDR2, and HCDR3, andwherein the light chain variable region is also composed of three CDRs,LCDR1, LCDR2, and LCDR3. The sequences of the CDRs are represented by:HCDR1 (SEQ ID NO:13), HCDR2 (SEQ ID NO:14), HCDR3 (SEQ ID NO:15), LCDR1(SEQ ID NO:16), LCDR2 (SEQ ID NO:17) and LCDR3 (SEQ ID NO:18). In someembodiments, the antibody further comprises a conjugated drug moiety.

In other embodiments, the isolated antibody is composed of a heavy chainvariable region, wherein the sequence of heavy chain variable region isencompassed by SEQ ID NO:19. In other embodiments, the isolated antibodyis composed of a light chain variable region, wherein the sequence ofthe light chain variable region is encompassed by SEQ ID NO:20. In someembodiments, the antibody further comprises a conjugated drug moiety.

In some embodiments, the isolated antibody is composed of a heavy chainvariable region, wherein the sequence of heavy chain variable region isencompassed by SEQ ID NO:19. In other embodiments, the isolated antibodyis composed of a light chain variable region, wherein the sequence ofthe light chain variable region is encompassed by SEQ ID NO:20. Thiscombination of heavy chain variable region and light chain variableregion is referred to as Ab19. In some embodiments, the antibody furthercomprises a conjugated drug moiety. In some embodiments, the antibodyalternatively further comprises a detectable label to facilitatediagnosis.

In some embodiments, the isolated antibody is composed of a heavy chainand a light chain, wherein the heavy chain sequence is encompassed bySEQ ID NO:21 and the light chain is encompassed by SEQ ID NO:22. In someembodiments, the antibody further comprises a conjugated drug moiety.

In some embodiments, an isolated nucleic acid encoding the heavy chainof SEQ ID NO:21 is provided. In other embodiments, an isolated nucleicacid encoding the light chain of SEQ ID NO:22 is provided.

In some embodiments, a host cell is provided, the host cell containingthe isolated nucleic acid encoding the heavy chain of SEQ ID NO:21 andthe isolated nucleic acid encoding the light chain of SEQ ID NO:22.

In some embodiments, a method of producing the antibody of the inventionis provided. The method encompassing culturing a host cell containingthe isolated nucleic acid encoding the heavy chain of SEQ ID NO:21 andthe isolated nucleic acid encoding the light chain of SEQ ID NO:22 underconditions wherein the isolated nucleic acid(s) are expressed and anantibody is produced. The drug moiety is then attached to the antibodyusing chemistries standard in the art.

In other embodiments, an isolated antibody specific for human CD38 (SEQID NO:1) and cynomolgus CD38 (SEQ ID NO:2) is described. This antibodyis composed of six CDRs, wherein each CDR of this antibody can differfrom SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, and SEQ ID NO:18 by 0, 1, or 2 amino acid substitutions.

In some embodiments, an isolated anti-CD38 antibody is provided thatbinds specifically to human CD38 (SEQ ID NO:1) and cynomolgus CD38 (SEQID NO:2), wherein the antibody binds to human CD38 with a KD of about10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or more and binds cynomolgus CD38 with a KD ofabout 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or more.

In some embodiments, antibodies that compete with Ab79 and/or Ab19 forbinding to human CD38 and/or cynomolgus CD38 are provided.

These and other embodiments, features and potential advantages willbecome apparent with reference to the following description anddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the CD38 Expression Profile on Lymphoid Lineage Cells.CD38 expression has been identified on pro-B cells (CD34⁺CD19⁺CD20⁻),activated B cells (CD19⁺CD20⁺), plasma cells (CD138⁺CD19⁻CD20⁻),activated CD4⁺ and CD8⁺ T cells, NKT cells (CD3⁺ CD56⁺) and NK cells(CD56⁺CD16⁺). In addition, CD38 expression is found on lymphoidprogenitor cells (CD34⁺CD45RA⁺CD10⁺CD19⁻) but not the lymphoid stemcell.

FIG. 2 shows the heavy and light chain sequences of Ab79 and Ab19.

FIG. 3 depicts the sequences of human and cynomolgus CD38.

FIG. 4 shows detection of Ab79 binding to normal tissues byimmunofluorescence. (A) normal human colon stained with Ab79 (B) normalhuman colon stained with irrelevant control antibody Palivizumab (C)light microscopy of normal human colon counterstained with hematoxylin(D) normal human prostate stained with Ab79 (E) normal human prostatestained with irrelevant control antibody Palivizumab (F) lightmicroscopy of normal human prostate counterstained with hematoxylin (G)normal human lymph node stained with Ab79 (H) light microscopy of normalhuman lymph node counterstained with hematoxylin

FIG. 5 shows detection of Ab79 binding to bone marrow samples fromMultiple Myeloma (MM) patients. (A) Immunofluorescent Ab79 staining ofnormal bone marrow (B) Representative immunofluorescent Ab79 staining ofbone marrow from a MM patient. The epitope recognized by Ab79 wasstrongly expressed in approximately 50% or more of the cells in allmultiple myeloma samples examined whereas in normal bone marrowapproximately 10% or less of the cells.

FIG. 6 depicts the binding of Ab79 to various cell lines as detected byimmunofluorescence. (A) MOLP-8 (B) Daudi (C) RPMI (D) MCF7.

FIG. 7 shows FACS analysis of Ab79 expression on cells derived from thebone marrow of a Multiple Myeloma patient (A) and PBMCs from a patientwith Chronic Lymphocytic Leukemia (gated on CD5⁺ cells) (B).

FIG. 8 depicts an in vivo dose response study comparing Ab19, Ab79, andbenchmark antibodies BM1 and BM2.

FIG. 9 depicts visualization of lymphoma dissemination in mice treatedwith Ab19, Ab79, and benchmark antibodies BM1 and BM2.

FIG. 10 depicts the binding of Ab79 to CD38 based on X-raycrystallography data.

FIGS. 11, 11A, 11B, 11C, 11D, and 11E depict a number of different ADCembodiments. As described herein, the linker moieties may change,including the composition of the amino acids, the self-immolativelinkers, etc.

FIG. 12 shows the epitopes of human CD38 that bind to each of theantibodies, Benchmark 1 and 2, Ab19 and Ab79.

FIG. 13 depicts the structure of a preferred linker/drug combination. Aswill be appreciated by those in the art, the TSF79 (e.g. Ab79) antibodydepicted herein can be switched to the AB19 antibody. Similarly, asdiscussed herein, any number of additional drug/linker combinations canbe used, in addition to the auristatin E derivative shown herein.

FIG. 14 depicts the percentage change in cell numbers in cyno monkeys at24 hours after dosing.

FIG. 15 shows the recovery of depletion after a single dose of Ab79.

DETAILED DESCRIPTION OF THE INVENTION Overview

The extracellular domain of CD38 has been shown to possess bifunctionalenzyme activity, having both ADP-ribosyl cyclase as well as ADP-ribosylhydrolase activities. Thus, CD38 can catalyze the conversion of NAD⁺ tocADPR (cyclase) and can further hydrolyze it to ADP-ribose (hydrolase).cADPR is involved in the mobilization of calcium from intracellularstores which is a second messenger activity important for cellularproliferation, differentiation, and apoptosis.

Increased expression of CD38 has been documented in a variety ofdiseases of hematopoietic origin and has been described as a negativeprognostic marker in chronic lymphoblastic leukemia. Such diseasesinclude but are not restricted to, multiple myeloma (Jackson et al.(1988)), chronic lymphoblastic leukemia (Moribito et al. (2001), Jelineket al. (2001), Chevalier et al. (2002), Dürig et al. (2002)), B-cellchronic lymphocytic leukemia, acute lymphoblastic leukemia (Keyhani etal (2000)) including B-cell acute lymphocytic leukemia, Waldenstrommacroglobulinemia, primary systemic amyloidosis, mantle-cell lymphoma,pro-lymphocytic/myelocytic leukemia, acute myeloid leukemia (Keyhani etal. (1993)), chronic myeloid leukemia (Marinov et al. (1993)),follicular lymphoma, NK-cell leukemia and plasma-cell leukemia. As such,CD38 provides a useful target in the treatment of diseases of thehematopoietic system.

Several anti-CD38 antibodies are in clinical trials for the treatment ofCD38-associated cancers (herein referred to as “Benchmark 1 andBenchmark 2”). Accordingly, antibodies to CD38 with therapeutic effectand/or diagnostic applications are useful. The invention provides twodifferent anti-CD38 sets of CDRs that bind to different epitopes ofCD38, and which bind both human and cynomolguso forms of CD38, andantibodies that contain these CDRs. The antibodies further comprisedrug/linker conjugates as discussed herein.

One advantage, not seen in some of the anti-CD38 antibodies in clinicaltesting, is the ability to bind to cynomolgus CD38, as these primatesfind use in preclinical testing, and thus can lead to early evaluationof dosing, toxicity, efficacy, etc.

CD38 Proteins

Accordingly, the present invention provides isolated anti-CD38antibodies that specifically bind human CD38 protein (and, as describedbelow, additionally and preferably specifically bind primate CD38protein). As is known in the art, CD38 proteins are found in a number ofspecies. Of particular use in the present invention are antibodies thatbind to both the human and primate CD38 proteins, particularly primatesused in clinical testing, such as cynomolgus (Macaca fascicularis, Crabeating macaque, sometimes referred to herein as “cyno”) monkeys. By“human CD38” or “human CD38 antigen” refers to the protein of SEQ IDNO:1 or a functional fraction, such as an epitope, as defined herein. Ingeneral, CD38 possesses a short intracytoplasmic tail, a transmembranedomain, and an extracellular domain, in specific embodiments, theantibodies of the invention bind to the extracellular part of the CD38protein. By “cynomolgus CD38” herein is meant SEQ ID NO:2 which is 92%identical to human CD38.

Synonyms of CD38, include ADP ribosyl cyclase 1, cADPr hydrolase 1,Cd38-rs1, Cyclic ADP-ribose hydrolase 1, 1-19, and NIM-R5 antigen.

In some embodiments, the anti-CD38 Ab79 antibodies of the inventioninteract with CD38 at a number of amino acid residues including K121,F135, Q139, D141, M142, D202, V203, H205, Q236, E239, W241, S274, C275,K276, F284, C287, V288, K289, N290, P291, E292, D293. As outlinedherein, other antibodies that interact with these residues also find usein therapeutic and diagnostic applications.

In some embodiments, the anti-CD38 antibodies of the present inventionoptionally (and in some cases preferably) do not bind to other membersof the CD38 family such as CD157. For example, preferred embodimentsherein do not bind to human CD157 of SEQ ID NO:23 (Genbank accessionNP_004325).

Antibodies

The present invention provides anti-CD38 antibodies, generallytherapeutic and/or diagnostic antibodies as described herein. Antibodiesthat find use in the present invention can take on a number of formatsas described herein, including traditional antibodies as well asantibody derivatives, fragments and mimetics, described below.Essentially, the invention provides antibody structures that contain aset of 6 CDRs as defined herein (including small numbers of amino acidchanges as described below).

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. Heavy chains are classifiedas mu, delta, gamma, alpha, or epsilon, and define the antibody'sisotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has severalsubclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4.IgM has subclasses, including, but not limited to, IgM1 and IgM2. Thus,“isotype” as used herein is meant any of the subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions. The known human immunoglobulin isotypes areIgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE. It shouldbe understood that therapeutic antibodies can also comprise hybrids ofisotypes and/or subclasses.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition. In the variable region, three loops are gathered for eachof the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5^(th) Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) (e.g, Kabat et al.,supra (1991)), with the EU number system used for the Fc region.

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.For example, as shown herein, the two different antibodies referred toherein as “Ab19” and Ab79” bind to different epitopes on the CD38molecule.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning”, as outlined in theExamples XRay crystallography studies as shown in the Examples hasidentified the amino acid residues that bind to the antibodies both ofthe invention (including Ab19 and Ab79) and the prior art (Benchmark 1and Benchmark 2), as shown in FIG. 12.

In the present invention, Ab79 as outlined in the Examples, interactswith a number of amino acid residues of CD38 including K121, F135, Q139,D141, M142, E239, W241, S274, C275, K276, F284, V288, K289, N290, P291,E292 and D293. It should be noted that these residues are identical inboth human and cyan monkeys, with the exception that S274 is actuallyF274 in cyan. These residues may represent the immunodominant eptitopeand/or residues within the footprint of the specifically antigen bindingpeptide.

In the present invention, Ab19 binds to a different epitope, includingG91, E103, E1034, D105, Q107, M110, K111, T114, Q115, T148, V192, R194,R195, F196, A199, H228, N229, Q231, E233 and K234. It should be notedthat these residues are identical in both human and cyan monkeys, withthe exception that M110 is V110 in cyan and A199 is T199 in cyan.

Thus, in some embodiments, antibodies that compete with AB79 and Ab19 bybinding at either of these epitopes can be used to treat autoimmunediseases. It should be noted that Ab79 and BM1 have some overlap; thusantibodies that compete with Ab79 and are not BM1 find use in thepresent invention.

Thus, the present invention provides antibodies that bind to both humanand cyan CD38 and interact with at least 80%, 90%, 95% or 98% of theseresidues. Stated differently, the surface area of the interaction zoneis no more than the area of these residues.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5^(th)edition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230.

Of particular interest in the present invention are the Fc regions. By“Fc” or “Fc region” or “Fc domain” as used herein is meant thepolypeptide comprising the constant region of an antibody excluding thefirst constant region immunoglobulin domain and in some cases, part ofthe hinge. Thus Fc refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, the last three constant regionimmunoglobulin domains of IgE and IgM, and the flexible hinge N-terminalto these domains. For IgA and IgM, Fc may include the J chain. For IgG,the Fc domain comprises immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3)and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to include residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. In some embodiments, as is more fully described below, aminoacid modifications are made to the Fc region, for example to alterbinding to one or more FcγR receptors or to the FcRn receptor.

In some embodiments, the antibodies are full length. By “full lengthantibody” herein is meant the structure that constitutes the naturalbiological form of an antibody, including variable and constant regions,including one or more modifications as outlined herein.

Alternatively, the antibodies can be a variety of structures, including,but not limited to, antibody fragments, monoclonal antibodies,bispecific antibodies, minibodies, domain antibodies, syntheticantibodies (sometimes referred to herein as “antibody mimetics”),chimeric antibodies, humanized antibodies, antibody fusions (sometimesreferred to as “antibody conjugates”), and fragments of each,respectively. Structures that still rely

In one embodiment, the antibody is an antibody fragment. Specificantibody fragments include, but are not limited to, (i) the Fab fragmentconsisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) the Fv fragment consistingof the VL and VH domains of a single antibody; (iv) the dAb fragment(Ward et al., 1989, Nature 341:544-546, entirely incorporated byreference) which consists of a single variable, (v) isolated CDRregions, (vi) F(ab′)2 fragments, a bivalent fragment comprising twolinked Fab fragments (vii) single chain Fv molecules (scFv), wherein aVH domain and a VL domain are linked by a peptide linker which allowsthe two domains to associate to form an antigen binding site (Bird etal., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:5879-5883, entirely incorporated by reference), (viii)bispecific single chain Fv (WO 03/11161, hereby incorporated byreference) and (ix) “diabodies” or “triabodies”, multivalent ormultispecific fragments constructed by gene fusion (Tomlinson et. al.,2000, Methods Enzymol. 326:461-479; WO94/13804; Holliger et al., 1993,Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448, all entirely incorporated byreference).

Chimeric and Humanized Antibodies

In some embodiments, the antibody can be a mixture from differentspecies, e.g. a chimeric antibody and/or a humanized antibody. That is,in the present invention, the CDR sets can be used with framework andconstant regions other than those specifically described by sequenceherein.

In general, both “chimeric antibodies” and “humanized antibodies” referto antibodies that combine regions from more than one species. Forexample, “chimeric antibodies” traditionally comprise variable region(s)from a mouse (or rat, in some cases) and the constant region(s) from ahuman. “Humanized antibodies” generally refer to non-human antibodiesthat have had the variable-domain framework regions swapped forsequences found in human antibodies. Generally, in a humanized antibody,the entire antibody, except the CDRs, is encoded by a polynucleotide ofhuman origin or is identical to such an antibody except within its CDRs.The CDRs, some or all of which are encoded by nucleic acids originatingin a non-human organism, are grafted into the beta-sheet framework of ahuman antibody variable region to create an antibody, the specificity ofwhich is determined by the engrafted CDRs. The creation of suchantibodies is described in, e.g., WO 92/11018, Jones, 1986, Nature321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536, all entirelyincorporated by reference. “Backmutation” of selected acceptor frameworkresidues to the corresponding donor residues is often required to regainaffinity that is lost in the initial grafted construct (U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; 5,859,205;5,821,337; 6,054,297; 6,407,213, all entirely incorporated byreference). The humanized antibody optimally also will comprise at leasta portion of an immunoglobulin constant region, typically that of ahuman immunoglobulin, and thus will typically comprise a human Fcregion. Humanized antibodies can also be generated using mice with agenetically engineered immune system. Roque et al., 2004, Biotechnol.Prog. 20:639-654, entirely incorporated by reference. A variety oftechniques and methods for humanizing and reshaping non-human antibodiesare well known in the art (See Tsurushita & Vasquez, 2004, Humanizationof Monoclonal Antibodies, Molecular Biology of B Cells, 533-545,Elsevier Science (USA), and references cited therein, all entirelyincorporated by reference). Humanization methods include but are notlimited to methods described in Jones et al., 1986, Nature 321:522-525;Riechmann et al., 1988; Nature 332:323-329; Verhoeyen et al., 1988,Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA86:10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al.,1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al., 1997, Cancer Res.57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8, all entirelyincorporated by reference. Humanization or other methods of reducing theimmunogenicity of nonhuman antibody variable regions may includeresurfacing methods, as described for example in Roguska et al., 1994,Proc. Natl. Acad. Sci. USA 91:969-973, entirely incorporated byreference. In one embodiment, the parent antibody has been affinitymatured, as is known in the art. Structure-based methods may be employedfor humanization and affinity maturation, for example as described inU.S. Ser. No. 11/004,590. Selection based methods may be employed tohumanize and/or affinity mature antibody variable regions, including butnot limited to methods described in Wu et al., 1999, J. Mol. Biol.294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684;Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al.,1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003,Protein Engineering 16(10):753-759, all entirely incorporated byreference. Other humanization methods may involve the grafting of onlyparts of the CDRs, including but not limited to methods described inU.S. Ser. No. 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125;De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all entirelyincorporated by reference.

In one embodiment, the antibodies of the invention can be multispecificantibodies, and notably bispecific antibodies, also sometimes referredto as “diabodies”. These are antibodies that bind to two (or more)different antigens, or different epitopes on the same antigen. Diabodiescan be manufactured in a variety of ways known in the art (Holliger andWinter, 1993, Current Opinion Biotechnol. 4:446-449, entirelyincorporated by reference), e.g., prepared chemically or from hybridhybridomas.

In one embodiment, the antibody is a minibody. Minibodies are minimizedantibody-like proteins comprising a scFv joined to a CH3 domain. Hu etal., 1996, Cancer Res. 56:3055-3061, entirely incorporated by reference.In some cases, the scFv can be joined to the Fc region, and may includesome or the entire hinge region.

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities. For instance, an isolated antibodythat specifically binds to CD38 is substantially free of antibodies thatspecifically bind antigens other than CD38.

An isolated antibody that specifically binds to an epitope, isoform orvariant of human CD38 or cynomolgus CD38 may, however, havecross-reactivity to other related antigens, for instance from otherspecies, such as CD38 species homologs. Moreover, an isolated antibodymay be substantially free of other cellular material and/or chemicals.

Isolated monoclonal antibodies, having different specificities, can becombined in a well defined composition. Thus for example the Ab79 andAb19 can be combined in a single formulation, if desired.

The anti-CD38 antibodies of the present invention specifically bind CD38ligands (e.g. the human and cynomolgus CD38 proteins of SEQ ID NOs:1 and2. “Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10⁻⁴ M, at least about 10⁻⁵ M, at least about10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M, at least about10⁻⁹M, alternatively at least about 10⁻¹⁰ M, at least about 10⁻¹¹ M, atleast about 10⁻¹² M, or greater, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction.

Antibody Modifications

The present invention further provides variant antibodies. That is,there are a number of modifications that can be made to the antibodiesof the invention, including, but not limited to, amino acidmodifications in the CDRs (affinity maturation), amino acidmodifications in the Fc region, glycosylation variants, covalentmodifications of other types, etc.

By “variant” herein is meant a polypeptide sequence that differs fromthat of a parent polypeptide by virtue of at least one amino acidmodification. Amino acid modifications can include substitutions,insertions and deletions, with the former being preferred in many cases.

In general, variants can include any number of modifications, as long asthe function of the protein is still present, as described herein. Thatis, in the case of amino acid variants generated with the CDRs of eitherAb79 or Ab19, for example, the antibody should still specifically bindto both human and cynomolgus CD38. Similarly, if amino acid variants aregenerated with the Fc region, for example, the variant antibodies shouldmaintain the required receptor binding functions for the particularapplication or indication of the antibody.

However, in general, from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions are generally utilized as often the goal is to alterfunction with a minimal number of modifications. In some cases, thereare from 1 to 5 modifications, with from 1-2, 1-3 and 1-4 also findinguse in many embodiments.

It should be noted that the number of amino acid modifications may bewithin functional domains: for example, it may be desirable to have from1-5 modifications in the Fc region of wild-type or engineered proteins,as well as from 1 to 5 modifications in the Fv region, for example. Avariant polypeptide sequence will preferably possess at least about 80%,85%, 90%, 95% or up to 98 or 99% identity to the parent sequences (e.g.the variable regions, the constant regions, and/or the heavy and lightchain sequences for Ab79 and/or Ab19). It should be noted that dependingon the size of the sequence, the percent identity will depend on thenumber of amino acids.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with another amino acid. For example, thesubstitution S100A refers to a variant polypeptide in which the serineat position 100 is replaced with alanine. By “amino acid insertion” or“insertion” as used herein is meant the addition of an amino acid at aparticular position in a parent polypeptide sequence. By “amino aciddeletion” or “deletion” as used herein is meant the removal of an aminoacid at a particular position in a parent polypeptide sequence.

By “parent polypeptide”, “parent protein”, “precursor polypeptide”, or“precursor protein” as used herein is meant an unmodified polypeptidethat is subsequently modified to generate a variant. In general, theparent polypeptides herein are Ab79 and Ab19. Parent polypeptide mayrefer to the polypeptide itself, compositions that comprise the parentpolypeptide, or the amino acid sequence that encodes it. Accordingly, by“parent Fc polypeptide” as used herein is meant an Fc polypeptide thatis modified to generate a variant, and by “parent antibody” as usedherein is meant an antibody that is modified to generate a variantantibody.

By “wild type” or “WT” or “native” herein is meant an amino acidsequence or a nucleotide sequence that is found in nature, includingallelic variations. A WT protein, polypeptide, antibody, immunoglobulin,IgG, etc. has an amino acid sequence or a nucleotide sequence that hasnot been intentionally modified.

By “variant Fc region” herein is meant an Fc sequence that differs fromthat of a wild-type Fc sequence by virtue of at least one amino acidmodification. Fc variant may refer to the Fc polypeptide itself,compositions comprising the Fc variant polypeptide, or the amino acidsequence.

In some embodiments, one or more amino acid modifications are made inone or more of the CDRs of the antibody (either Ab79 or Ab19). Ingeneral, only 1 or 2 or 3 amino acids are substituted in any single CDR,and generally no more than from 4, 5, 6, 7, 8 9 or 10 changes are madewithin a set of CDRs. However, it should be appreciated that anycombination of no substitutions, 1, 2 or 3 substitutions in any CDR canbe independently and optionally combined with any other substitution.

In some cases, amino acid modifications in the CDRs are referred to as“affinity maturation”. An “affinity matured” antibody is one having oneor more alteration(s) in one or more CDRs which results in animprovement in the affinity of the antibody for antigen, compared to aparent antibody which does not possess those alteration(s). In somecases, although rare, it may be desirable to decrease the affinity of anantibody to its antigen, but this is generally not preferred.

Affinity maturation can be done to increase the binding affinity of theantibody for the antigen by at least about 10% to 50-100-150% or more,or from 1 to 5 fold as compared to the “parent” antibody. Preferredaffinity matured antibodies will have nanomolar or even picomolaraffinities for the target antigen. Affinity matured antibodies areproduced by known procedures. See, for example, Marks et al., 1992,Biotechnology 10:779-783 that describes affinity maturation by variableheavy chain (VH) and variable light chain (VL) domain shuffling. Randommutagenesis of CDR and/or framework residues is described in: Barbas, etal. 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995,Gene 169:147-155; Yelton et al., 1995, J. Immunol. 155:1994-2004;Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al,1992, J. Mol. Biol. 226:889-896, for example.

Alternatively, amino acid modifications can be made in one or more ofthe CDRs of the antibodies of the invention that are “silent”, e.g. thatdo not significantly alter the affinity of the antibody for the antigen.These can be made for a number of reasons, including optimizingexpression (as can be done for the nucleic acids encoding the antibodiesof the invention).

Thus, included within the definition of the CDRs and antibodies of theinvention are variant CDRs and antibodies; that is, the antibodies ofthe invention can include amino acid modifications in one or more of theCDRs of Ab79 and Ab19. In addition, as outlined below, amino acidmodifications can also independently and optionally be made in anyregion outside the CDRs, including framework and constant regions.

In some embodiments, variant antibodies of Ab79 and Ab19 that arespecific for human CD38 (SEQ ID NO:1) and cynomolgus CD38 (SEQ ID NO:2)is described. This antibody is composed of six CDRs, wherein each CDR ofthis antibody can differ from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, and SEQ ID NO:8 by 0, 1, or 2 amino acidsubstitutions. In other embodiments, the variant anti-CD38 antibody iscomposed of six CDRs, wherein each CDR of this antibody can differ fromSEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,and SEQ ID NO:18 by 0, 1, or 2 amino acid substitutions.

In some embodiments, the anti-CD38 antibodies of the invention arecomposed of a variant Fc domain. As is known in the art, the Fc regionof an antibody interacts with a number of Fc receptors and ligands,imparting an array of important functional capabilities referred to aseffector functions. These Fc receptors include, but are not limited to,(in humans) FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc;FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 andR131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; andFcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158and F158, correlated to antibody-dependent cell cytotoxicity (ADCC)) andFcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), FcRn (theneonatal receptor), C1q (complement protein involved in complementdependent cytotoxicity (CDC)) and FcRn (the neonatal receptor involvedin serum half-life). Suitable modifications can be made at one or morepositions as is generally outlined, for example in U.S. patentapplication Ser. No. 11/841,654 and references cited therein, US2004/013210, US 2005/0054832, US 2006/0024298, US 2006/0121032, US2006/0235208, US 2007/0148170, U.S. Ser. No. 12/341,769, U.S. Pat. Nos.6,737,056, 7,670,600, 6,086,875 all of which are expressly incorporatedby reference in their entirety, and in particular for specific aminoacid substitutions that increase binding to Fc receptors.

In addition to the modifications outlined above, other modifications canbe made. For example, the molecules may be stabilized by theincorporation of disulphide bridges linking the VH and VL domains(Reiter et al., 1996, Nature Biotech. 14:1239-1245, entirelyincorporated by reference). In addition, there are a variety of covalentmodifications of antibodies that can be made as outlined below.

Covalent modifications of antibodies are included within the scope ofthis invention, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antibody are introduced into the molecule byreacting specific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesmay also be derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole and the like.

In addition, modifications at cysteines are particularly useful inantibody-drug conjugate (ADC) applications, further described below. Insome embodiments, the constant region of the antibodies can beengineered to contain one or more cysteines that are particularly “thiolreactive”, so as to allow more specific and controlled placement of thedrug moiety. See for example U.S. Pat. No. 7,521,541, incorporated byreference in its entirety herein.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using 125I or 131I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantibodies to a water-insoluble support matrix or surface for use in avariety of methods, in addition to methods described below. Commonlyused crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis (succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such ascynomolgusogen bromide-activated carbohydrates and the reactivesubstrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128;4,247,642; 4,229,537; and 4,330,440, all entirely incorporated byreference, are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983],entirely incorporated by reference), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

In addition, as will be appreciated by those in the art, labels(including fluorescent, enzymatic, magnetic, radioactive, etc. can allbe added to the antibodies (as well as the other compositions of theinvention).

Glycosylation

Another type of covalent modification is alterations in glycosylation.In another embodiment, the antibodies disclosed herein can be modifiedto include one or more engineered glycoforms. By “engineered glycoform”as used herein is meant a carbohydrate composition that is covalentlyattached to the antibody, wherein said carbohydrate composition differschemically from that of a parent antibody. Engineered glycoforms may beuseful for a variety of purposes, including but not limited to enhancingor reducing effector function. A preferred form of engineered glycoformis afucosylation, which has been shown to be correlated to an increasein ADCC function, presumably through tighter binding to the FcγRIIIareceptor. In this context, “afucosylation” means that the majority ofthe antibody produced in the host cells is substantially devoid offucose, e.g. 90-95-98% of the generated antibodies do not haveappreciable fucose as a component of the carbohydrate moiety of theantibody (generally attached at N297 in the Fc region). Definedfunctionally, afucosylated antibodies generally exhibit at least a 50%or higher affinity to the FcγRIIIa receptor.

Engineered glycoforms may be generated by a variety of methods known inthe art (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies et al.,2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S.Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929;PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO02/30954A1, all entirely incorporated by reference; (Potelligent®technology [Biowa, Inc., Princeton, N.J.]; GlycoMAb® glycosylationengineering technology [Glycart Biotechnology AG, Zurich, Switzerland]).Many of these techniques are based on controlling the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, for example by expressing an IgG in variousorganisms or cell lines, engineered or otherwise (for example Lec-13 CHOcells or rat hybridoma YB2/0 cells, by regulating enzymes involved inthe glycosylation pathway (for example FUT8 [a1,6-fucosyltranserase]and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or bymodifying carbohydrate(s) after the IgG has been expressed. For example,the “sugar engineered antibody” or “SEA technology” of Seattle Geneticsfunctions by adding modified saccharides that inhibit fucosylationduring production; see for example 20090317869, hereby incorporated byreference in its entirety. Engineered glycoform typically refers to thedifferent carbohydrate or oligosaccharide; thus an antibody can includean engineered glycoform.

Alternatively, engineered glycoform may refer to the IgG variant thatcomprises the different carbohydrate or oligosaccharide. As is known inthe art, glycosylation patterns can depend on both the sequence of theprotein (e.g., the presence or absence of particular glycosylation aminoacid residues, discussed below), or the host cell or organism in whichthe protein is produced. Particular expression systems are discussedbelow.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tri-peptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thestarting sequence (for O-linked glycosylation sites). For ease, theantibody amino acid sequence is preferably altered through changes atthe DNA level, particularly by mutating the DNA encoding the targetpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody is by chemical or enzymatic coupling of glycosides to theprotein. These procedures are advantageous in that they do not requireproduction of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 and in Aplin andWriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both entirelyincorporated by reference.

Removal of carbohydrate moieties present on the starting antibody (e.g.post-translationally) may be accomplished chemically or enzymatically.Chemical deglycosylation requires exposure of the protein to thecompound trifluoromethanesulfonic acid, or an equivalent compound. Thistreatment results in the cleavage of most or all sugars except thelinking sugar (N-acetylglucosamine or N-acetylgalactosamine), whileleaving the polypeptide intact. Chemical deglycosylation is described byHakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge etal., 1981, Anal. Biochem. 118:131, both entirely incorporated byreference. Enzymatic cleavage of carbohydrate moieties on polypeptidescan be achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., 1987, Meth. Enzymol. 138:350, entirelyincorporated by reference. Glycosylation at potential glycosylationsites may be prevented by the use of the compound tunicamycin asdescribed by Duskin et al., 1982, J. Biol. Chem. 257:3105, entirelyincorporated by reference. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the antibody comprises linkingthe antibody to various nonproteinaceous polymers, including, but notlimited to, various polyols such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in, for example,2005-2006 PEG Catalog from Nektar Therapeutics (available at the Nektarwebsite) U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337, all entirely incorporated by reference. Inaddition, as is known in the art, amino acid substitutions may be madein various positions within the antibody to facilitate the addition ofpolymers such as PEG. See for example, U.S. Publication No.2005/0114037A1, entirely incorporated by reference.

Specific CDR and Variable Region Embodiments

The present invention provides a number of antibodies each with aspecific set of CDRs (including, as outlined above, some amino acidsubstitutions). As outlined above, the antibodies can be defined by setsof 6 CDRs, by variable regions, or by full-length heavy and lightchains, including the constant regions. In addition, as outlined above,amino acid substitutions may also be made. In general, in the context ofchanges within CDRs, due to the relatively short length of the CDRs, theamino acid modifications are generally described in terms of the numberof amino acid modifications that may be made. While this is alsoapplicable to the discussion of the number of amino acid modificationsthat can be introduced in variable, constant or full length sequences,in addition to number of changes, it is also appropriate to define thesechanges in terms of the “% identity”. Thus, as described herein,antibodies included within the invention are 80, 85, 90, 95, 98 or 99%identical to the SEQ ID NOs listed herein.

In the context of the Ab79 antibody, the set of CDRs is as follows: thethree CDRs of the heavy chain encompass HCDR1 SEQ ID NO:3 (HCDR1), SEQID NO:4 (HCDR2), and SEQ ID NO:5 (HCDR3), and the three CDRs of thelight chain encompass SEQ ID NO:6 (LCDR1), SEQ ID NO:7 (LCDR2), and SEQID NO:8 (LCDR3).

In the context of Ab19, the set of CDRs is as follows: HCDR1 (SEQ IDNO:13), HCDR2 (SEQ ID NO:14), and HCDR3 (SEQ ID NO:15), and LCDR1 (SEQID NO:16), LCDR2 (SEQ ID NO:17), and LCDR3 (SEQ ID NO:18).

Specifically excluded from the present invention are the antibodies ofSEQ ID NOs.: 24 and 25 (the heavy and light chains of Benchmark 1) andSEQ ID NOs.: 26 and 27 (the heavy and light chains of Benchmark 2). Itshould be noted that these antibodies are not cross reactive withcynomolgus CD38, discussed below.

The antibodies of the invention are cross reactive with human andcynomolgus CD38 and are thus species cross-reactive antibodies. A“species cross-reactive antibody” is an antibody that has a bindingaffinity for an antigen from a first mammalian species that is nearlythe same as the binding affinity for a homologue of that antigen from asecond mammalian species. Species cross-reactivity can be expressed, forexample, as a ratio of the KD of an antibody for an antigen of the firstmammalian species over the KD of the same antibody for the homologue ofthat antigen from a second mammalian species wherein the ratio is 1.1,1.2, 1.3, 1.4, 1.5, 2, 5, 10, 15, up to 20. Alternatively oradditionally, an antibody is “species cross reactive” when it showstherapeutic or diagnostic efficacy when administered to the secondspecies. Thus, in the present case, the antibodies of the invention arecross reactive with cynomolgus CD38, show preclinical efficacy whenadministered to cynomolgus primates and thus are considered crossreactive.

In some embodiments, antibodies that compete with the antibodies of theinvention (for example, with Ab79 and/or Ab19) for binding to human CD38and/or cynomolgus CD38 are provided, but are not either BM1 or BM2 areincluded. Competition for binding to CD38 or a portion of CD38 by two ormore anti-CD38 antibodies may be determined by any suitable technique,as is known in the art.

Competition in the context of the present invention refers to anydetectably significant reduction in the propensity of an antibody of theinvention (e.g. Ab79 or Ab19) to bind its particular binding partner,e.g. CD38, in the presence of the test compound. Typically, competitionmeans an at least about 10-100% reduction in the binding of an antibodyof the invention to CD38 in the presence of the competitor, as measuredby standard techniques such as ELISA or Biacore® assays. Thus, forexample, it is possible to set criteria for competitiveness wherein atleast about 10% relative inhibition is detected; at least about 15%relative inhibition is detected; or at least about 20% relativeinhibition is detected before an antibody is considered sufficientlycompetitive. In cases where epitopes belonging to competing antibodiesare closely located in an antigen, competition may be marked by greaterthan about 40% relative inhibition of CD38 binding (e.g., at least about45% inhibition, such as at least about 50% inhibition, for instance atleast about 55% inhibition, such as at least about 60% inhibition, forinstance at least about 65% inhibition, such as at least about 70%inhibition, for instance at least about 75% inhibition, such as at leastabout 80% inhibition, for instance at least about 85% inhibition, suchas at least about 90% inhibition, for instance at least about 95%inhibition, or higher level of relative inhibition).

In some cases, one or more of the components of the competitive bindingassays are labeled, as discussed below in the context of diagnosticapplications.

It may also be the case that competition may exist between anti-CD38antibodies with respect to more than one of CD38 epitope, and/or aportion of CD38, e.g. in a context where the antibody-binding propertiesof a particular region of CD38 are retained in fragments thereof, suchas in the case of a well-presented linear epitope located in varioustested fragments or a conformational epitope that is presented insufficiently large CD38 fragments as well as in CD38.

Assessing competition typically involves an evaluation of relativeinhibitory binding using an antibody of the invention, CD38 (eitherhuman or cynomolgus or both), and the test molecule. Test molecules caninclude any molecule, including other antibodies, small molecules,peptides, etc. The compounds are mixed in amounts that are sufficient tomake a comparison that imparts information about the selectivity and/orspecificity of the molecules at issue with respect to the other presentmolecules.

The amounts of test compound, CD38 and antibodies of the invention maybe varied. For instance, for ELISA assessments about 5-50 μg (e.g.,about 10-50 about 20-50 about 5-20 about 10-20 etc.) of the anti-CD38antibody and/or CD38 targets are required to assess whether competitionexists. Conditions also should be suitable for binding. Typically,physiological or near-physiological conditions (e.g., temperatures ofabout 20-40° C., pH of about 7-8, etc.) are suitable for anti-CD38:CD38binding.

Often competition is marked by a significantly greater relativeinhibition than about 5% as determined by ELISA and/or FACS analysis. Itmay be desirable to set a higher threshold of relative inhibition as acriteria/determinant of what is a suitable level of competition in aparticular context (e.g., where the competition analysis is used toselect or screen for new antibodies designed with the intended functionof blocking the binding of another peptide or molecule binding to CD38(e.g., the natural binding partners of CD38 such as CD31, also calledCD31 antigen, EndoCAM, GPIIA, PECAM-1, platelet/endothelial celladhesion molecule or naturally occurring anti-CD38 antibody).

In some embodiments, the anti-CD38 antibody of the present inventionspecifically binds to one or more residues or regions in CD38 but alsodoes not cross-react with other proteins with homology to CD38, such asBST-1 (bone marrow stromal cell antigen-1) and Mo5, also called CD157.

Typically, a lack of cross-reactivity means less than about 5% relativecompetitive inhibition between the molecules when assessed by ELISAand/or FACS analysis using sufficient amounts of the molecules undersuitable assay conditions.

The disclosed antibodies may find use in blocking a ligand-receptorinteraction or inhibiting receptor component interaction. The anti-CD38antibodies of the invention may be “blocking” or “neutralizing.” A“neutralizing antibody” is intended to refer to an antibody whosebinding to CD38 results in inhibition of the biological activity ofCD38, for example its capacity to interact with ligands, enzymaticactivity, and/or signaling capacity. Inhibition of the biologicalactivity of CD38 can be assessed by one or more of several standard invitro or in vivo assays known in the art (see examples below).

“Inhibits binding” or “blocks binding” (for instance when referring toinhibition/blocking of binding of a CD38 binding partner to CD38)encompass both partial and complete inhibition/blocking. Theinhibition/blocking of binding of a CD38 binding partner to CD38 mayreduce or alter the normal level or type of cell signaling that occurswhen a CD38 binding partner binds to CD38 without inhibition orblocking. Inhibition and blocking are also intended to include anymeasurable decrease in the binding affinity of a CD38 binding partner toCD38 when in contact with an anti-CD38 antibody, as compared to theligand not in contact with an anti-CD38 antibody, for instance ablocking of binding of a CD38 binding partner to CD38 by at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

The disclosed anti-CD38 antibodies may also inhibit cell growth.“Inhibits growth” includes any measurable decrease in the cell growthwhen contacted with a an anti-CD38 antibody, as compared to the growthof the same cells not in contact with an anti-CD38 antibody, forinstance an inhibition of growth of a cell culture by at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

Methods for Producing the Antibodies of the Invention

The present invention further provides methods for producing thedisclosed anti-CD38 antibodies. These methods encompass culturing a hostcell containing isolated nucleic acid(s) encoding the antibodies of theinvention. As will be appreciated by those in the art, this can be donein a variety of ways, depending on the nature of the antibody. In someembodiments, in the case where the antibodies of the invention are fulllength traditional antibodies, for example, a heavy chain variableregion and a light chain variable region under conditions such that anantibody is produced and can be isolated.

In general, nucleic acids are provided that encode the antibodies of theinvention. Such polynucleotides encode for both the variable andconstant regions of each of the heavy and light chains, although othercombinations are also contemplated by the present invention inaccordance with the compositions described herein. The present inventionalso contemplates oligonucleotide fragments derived from the disclosedpolynucleotides and nucleic acid sequences complementary to thesepolynucleotides.

The polynucleotides can be in the form of RNA or DNA. Polynucleotides inthe form of DNA, cDNA, genomic DNA, nucleic acid analogs, and syntheticDNA are within the scope of the present invention. The DNA may bedouble-stranded or single-stranded, and if single stranded, may be thecoding (sense) strand or non-coding (anti-sense) strand. The codingsequence that encodes the polypeptide may be identical to the codingsequence provided herein or may be a different coding sequence, whichsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same polypeptides as the DNA provided herein.

In some embodiments, nucleic acid(s) encoding the antibodies of theinvention are incorporated into expression vectors, which can beextrachromosomal or designed to integrate into the genome of the hostcell into which it is introduced. Expression vectors can contain anynumber of appropriate regulatory sequences (including, but not limitedto, transcriptional and translational control sequences, promoters,ribosomal binding sites, enhancers, origins of replication, etc.) orother components (selection genes, etc.), all of which are operablylinked as is well known in the art. In some cases two nucleic acids areused and each put into a different expression vector (e.g. heavy chainin a first expression vector, light chain in a second expressionvector), or alternatively they can be put in the same expression vector.It will be appreciated by those skilled in the art that the design ofthe expression vector(s), including the selection of regulatorysequences may depend on such factors as the choice of the host cell, thelevel of expression of protein desired, etc.

In general, the nucleic acids and/or expression can be introduced into asuitable host cell to create a recombinant host cell using any methodappropriate to the host cell selected (e.g., transformation,transfection, electroporation, infection), such that the nucleic acidmolecule(s) are operably linked to one or more expression controlelements (e.g., in a vector, in a construct created by processes in thecell, integrated into the host cell genome). The resulting recombinanthost cell can be maintained under conditions suitable for expression(e.g. in the presence of an inducer, in a suitable non-human animal, insuitable culture media supplemented with appropriate salts, growthfactors, antibiotics, nutritional supplements, etc.), whereby theencoded polypeptide(s) are produced. In some cases, the heavy chains areproduced in one cell and the light chain in another.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC), Manassas, Va. including but not limitedto Chinese hamster ovary (CHO) cells, HEK 293 cells, NSO cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. Non-mammalian cells including but not limited to bacterial,yeast, insect, and plants can also be used to express recombinantantibodies. In some embodiments, the antibodies can be produced intransgenic animals such as cows or chickens.

General methods for antibody molecular biology, expression,purification, and screening are described, for example, in AntibodyEngineering, edited by Kontermann & Dubel, Springer, Heidelberg, 2001and 2010 Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689;Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; and Morrison, S.(1985) Science 229:1202.

Antibody drug conjugates as described herein are made as is known in theart, including techniques utilized by Seattle Genetics (see for exampleU.S. Pat. Nos. 8,067,546, 8,039,273, 7,989,434, 7,851,437, 7,837,980 and7,829,531, all of which are expressly incorporated in their entirety byreference, with particular reference to drugs, linkers and methods ofconjugation), Syntarga (see for example U.S. Pat. Nos. 7,705,045 and7,223,837, all of which are expressly incorporated in their entirety byreference, with particular reference to drugs, linkers and methods ofconjugation), Medarex (see for example U.S. Pat. Nos. 8,034,959,8,034,787, 7,968,586, 7,847,105, all of which are expressly incorporatedin their entirety by reference, with particular reference to drugs,linkers and methods of conjugation), and others well known in the art.

Applications and Indications

Once made, the antibodies of the invention find use in a variety ofapplications, including diagnosis of CD38-related diseases and treatmentthereof.

CD38 Related Conditions

In one aspect, the invention provides methods of treating a conditionassociated with proliferation of cells expressing CD38, comprisingadministering to a patient a pharmaceutically effective amount of adisclosed antibody. In certain embodiments, the condition is cancer, andin particular embodiments, the cancer is hematological cancer. In otherparticular embodiments, the condition is multiple myeloma, chroniclymphoblastic leukemia, chronic lymphocytic leukemia, plasma cellleukemia, acute myeloid leukemia, chronic myeloid leukemia, B-celllymphoma, or Burkitt's lymphoma.

It is known in the art that certain conditions are associated with cellsthat express CD38, and that certain conditions are associated with theoverexpression, high-density expression, or upregulated expression ofCD38 on the surfaces of cells. Whether a cell population expresses CD38or not can be determined by methods known in the art, for example flowcytometric determination of the percentage of cells in a givenpopulation that are labelled by an antibody that specifically binds CD38or immunohistochemical assays, as are generally described below fordiagnostic applications. For example, a population of cells in whichCD38 expression is detected in about 10-30% of the cells can be regardedas having weak positivity for CD38; and a population of cells in whichCD38 expression is detected in greater than about 30% of the cells canbe regarded as definite positivity for CD38 (as in Jackson et al.(1988), Clin. Exp. Immunol. 72: 351-356), though other criteria can beused to determine whether a population of cells expresses CD38. Densityof expression on the surfaces of cells can be determined using methodsknown in the art, such as, for example, flow cytometric measurement ofthe mean fluorescence intensity of cells that have been fluorescentlylabelled using antibodies that specifically bind CD38.

In some embodiments, the compositions and methods of the invention areapplied to a cancer such as a “hematologic cancer,” a term that refersto malignant neoplasms of blood-forming tissues and encompassesleukemia, lymphoma and multiple myeloma. Non-limiting examples ofconditions associated with CD38 expression include but are not limitedto, multiple myeloma (Jackson et al. (1988), Clin. Exp. Immunol. 72:351-356), B-cell chronic lymphocytic leukemia (B-CLL) Dürig et al.(2002), Leukemia 16: 30-5; Morabito et al. (2001), Leukemia Research 25:927-32; Marinov et al. (1993), Neoplasma 40(6): 355-8; and Jelinek etal. (2001), Br. J. Haematol. 115: 854-61), acute lymphoblastic leukemia(Keyhani et al. (1999), Leukemia Research 24: 153-9; and Marinov et al.(1993), Neoplasma 40(6): 355-8), chronic myeloid leukemia (Marinov etal. (1993), Neoplasma 40(6): 355-8), acute myeloid leukemia (Keyhani etal. (1999), Leukemia Research 24: 153-9), chronic lymphocytic leukemia(CLL), chronic myelogenous leukemia or chronic myeloid leukemia (CML),acute myelogenous leukemia or acute myeloid leukemia (AML), acutelymphocytic leukemia (ALL), hairy cell leukemia (HCL), myelodysplasticsyndromes (MDS) or chronic myelogenous leukemia (CML-BP) in blastic andall subtypes of these leukemias which are defined by morphological,histochemical and immunological techniques that are well known by thoseof skill in the art.

“Neoplasm” or “neoplastic condition” refers to a condition associatedwith proliferation of cells characterized by a loss of normal controlsthat results in one ore more symptoms including, unregulated growth,lack of differentiation, local tissue invasion, and metastasis.

In some embodiments of the invention, the hematologic cancer is aselected from the group of Chronic Lymphocytic Leukemia (CLL), ChronicMyelogenous Leukemia (CML), Acute Myelogenous Leukemia (AML), and AcuteLymphocytic Leukemia (ALL).

Furthermore, it is known in the art that CD38 expression is a prognosticindicator for patients with conditions such as, for example, B-cellchronic lymphocytic leukemia (Dürig et al. (2002), Leukemia 16: 30-5;and Morabito et al. (2001), Leukemia Research 25: 927-32) and acutemyelogenous leukemia (Keyhani et al. (1999), Leukemia Research 24:153-9).

CLL is the most common leukemia of adults in the Western world. CLLinvolves clonal expansion of mature-appearing lymphocytes involvinglymph nodes and other lymphoid tissues with progressive infiltration ofbone marrow and presence in the peripheral blood. The B-cell form(B-CLL) represents almost all cases.

B-CLL

B-CLL is an incurable disease characterized by a progressive increase ofanergic monoclonal B lineage cells that accumulate in the bone marrowand peripheral blood in a protracted fashion over many years. Theexpression of CD38 is regarded as an independent poor prognostic factorfor B-CLL. Hamblin et al., Blood 99:1023-9 (2002).

Today's standard therapy of B-CLL is palliative and is mainly carriedout with the cytostatic agent chlorambucil or fludarabine. When relapsesoccur, a combination therapy using fludarabine, cyclophosphamide incombination with rituximab (monoclonal antibody against CD20) or campath(monoclonal antibody against CD52) is often initiated. Thus, there is acritical unmet medical need for the treatment of B-CLL. In someembodiments, methods for treating B-CLL using the disclosed anti-CD38antibodies are provided (and, as outlined below, this may be done usingcombination therapies including optionally and independently any of theabove drugs).

B-CLL is characterized by two subtypes, indolent and aggressive. Theseclinical phenotypes correlate with the presence or absence of somaticmutations in the immunoglobulin heavy-chain variable region (IgVH) gene.As used herein, indolent B-CLL refers to a disorder in a subjects havingmutated IgVH gene and/or presenting with one or more clinical phenotypesassociated with indolent B-CLL. As used herein, the phrase aggressiveB-CLL refers to a disorder in a subject having unmutated IgVH and/orpresenting with one or more clinical phenotypes associated withaggressive B-CLL.

Multiple Myeloma

Multiple myeloma is a malignant disorder of the B cell lineagecharacterized by neoplastic proliferation of plasma cells in the bonemarrow. Current treatment regimens exhibit moderate response rates.However, only marginal changes in overall survival are observed and themedian survival is approximately 3 years. Thus, there is a criticalunmet medical need for the treatment of multiple myeloma. In someembodiments, methods for treating multiple myeloma using the disclosedantibodies are provided.

CD38 is highly expressed on plasma cells which are terminallydifferentiated B cells.

Proliferation of myeloma cells causes a variety of effects, includinglytic lesions (holes) in the bone, decreased red blood cell number,production of abnormal proteins (with attendant damage to the kidney,nerves, and other organs), reduced immune system function, and elevatedblood calcium levels (hypercalcemia).

Currently treatment options include chemotherapy, preferably associatedwhen possible with autologous stem cell transplantation (ASCT).

Monoclonal Gammopathy of Undetermined Significance and SmolderingMultiple Myeloma

In some embodiments, methods for treating monoclonal gammopathy usingthe disclosed antibodies are provided. In other embodiments, methods fortreating smoldering multiple myeloma using the disclosed antibodies areprovided.

Monoclonal gammopathy of undetermined significance (MGUS) and smolderingmultiple myeloma (SMM) are asymptomatic, pre-malignant disorderscharacterized by monoclonal plasma cell proliferation in the bone marrowand absence of end-organ damage.

Smoldering multiple myeloma (SMM) is an asymptomatic proliferativedisorder of plasma cells with a high risk of progression to symptomatic,or active multiple myeloma (N. Engl. J. Med. 356(25): 2582-2590 (2007)).

International consensus criteria defining SMM were adopted in 2003 andrequire that a patient have a M-protein level of >30 g/L and/or bonemarrow clonal plasma cells >10% (Br. J. Haematol. 121: 749-57 (2003)).The patient must have no organ or related tissue impairment, includingbone lesions or symptoms (Br. J. Haematol. 121: 749-57 (2003)).

Recent studies have identified two subsets of SMM; i) patients withevolving. disease and ii) patients with non-evolving disease (Br. J.Haematol. 121: 631-636 (2003)). International consensus criteriadefining MGUS require that a patient have a M-protein level of <30 g/L,bone marrow plasma cells <10% and the absence of organ or related tissueimpairment, including bone lesions or symptoms (Br. J. Haematol. 121:749-57 (2003)).

SMM resembles monoclonal gammopathy of undetermined significance (MGUS)as end-organ damage is absent (N. Engl. J. Med. 356(25): 2582-2590(2007)). Clinically, however, SMM is far more likely to progress toactive multiple myeloma or amyloidosis at 20 years (78% probability forSMM vs. 21% for MGUS) (N. Engl. J. Med. 356(25): 2582-2590 (2007)).

FIGS. 11, 11A, 11B, 11C, 11D and 11E depict a number of different ADCembodiments. As described herein, the linker moieties may change,including the composition of the amino acids, the self-immolativelinkers, etc.

Antibody-Drug Conjugates

In some embodiments, the anti-CD38 antibodies of the invention areconjugated with drugs to form antibody-drug conjugates (ADCs). Ingeneral, ADCs are used in oncology applications, where the use ofantibody-drug conjugates for the local delivery of cytotoxic orcytostatic agents allows for the targeted delivery of the drug moiety totumors, which can allow higher efficacy, lower toxicity, etc. Anoverview of this technology is provided in Ducry et al., BioconjugateChem., 21:5-13 (2010), Carter et al., Cancer J. 14(3):154 (2008) andSenter, Current Opin. Chem. Biol. 13:235-244 (2009), all of which arehereby incorporated by reference in their entirety

Thus the invention provides anti-CD38 antibodies conjugated to drugs.Generally, conjugation is done by covalent attachment to the antibody,as further described below, and generally relies on a linker, often apeptide linkage (which, as described below, may be designed to besensitive to cleavage by proteases at the target site or not). Inaddition, as described above, linkage of the linker-drug unit (LU-D) canbe done by attachment to cysteines within the antibody. As will beappreciated by those in the art, the number of drug moieties perantibody can change, depending on the conditions of the reaction, andcan vary from 1:1 to 10:1 drug:antibody. As will be appreciated by thosein the art, the actual number is an average.

Thus the invention provides anti-CD38 antibodies conjugated to drugs. Asdescribed below, the drug of the ADC can be any number of agents,including but not limited to cytotoxic agents such as chemotherapeuticagents, growth inhibitory agents, toxins (for example, an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (that is, a radioconjugate) areprovided. In other embodiments, the invention further provides methodsof using the ADCs.

Drugs for use in the present invention include cytotoxic drugs,particularly those which are used for cancer therapy. Such drugsinclude, in general, DNA damaging agents, anti-metabolites, naturalproducts and their analogs. Exemplary classes of cytotoxic agentsinclude the enzyme inhibitors such as dihydrofolate reductaseinhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNAcleavers, topoisomerase inhibitors, the anthracycline family of drugs,the vinca drugs, the mitomycins, the bleomycins, the cytotoxicnucleosides, the pteridine family of drugs, diynenes, thepodophyllotoxins, dolastatins, maytansinoids, differentiation inducers,and taxols.

Members of these classes include, for example, methotrexate,methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine,cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin,daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin,aminopterin, tallysomycin, podophyllotoxin and podophyllotoxinderivatives such as etoposide or etoposide phosphate, vinblastine,vincristine, vindesine, taxanes including taxol, taxotere retinoic acid,butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin,esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin,camptothecin, maytansinoids (including DM1), monomethylauristatin E(MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and theiranalogues.

Toxins may be used as antibody-toxin conjugates and include bacterialtoxins such as diphtheria toxin, plant toxins such as ricin, smallmolecule toxins such as geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342).Toxins may exert their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.

Conjugates of an anti-CD38 antibody and one or more small moleculetoxins, such as a maytansinoids, dolastatins, auristatins, atrichothecene, calicheamicin, and CC1065, and the derivatives of thesetoxins that have toxin activity, are contemplated.

Maytansinoids

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art, and can be isolated from natural sourcesaccording to known methods, produced using genetic engineeringtechniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol andmaytansinol analogues prepared synthetically according to known methods.As described below, drugs may be modified by the incorporation of afunctionally active group such as a thiol or amine group for conjugationto the antibody.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl) +/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared byacylation using acyl chlorides) and those having modifications at otherpositions

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H2S or P2S5);C-14-alkoxymethyl(demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.4,364,866) (prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol).

Of particular use are DM1 (disclosed in U.S. Pat. No. 5,208,020,incorporated by reference) and DM4 (disclosed in U.S. Pat. No.7,276,497, incorporated by reference). See also a number of additionalmaytansinoid derivatives and methods in U.S. Pat. No. 5,416,064,WO/01/24763, U.S. Pat. Nos. 7,303,749, 7,601,354, U.S. Ser. No.12/631,508, WO02/098883, U.S. Pat. Nos. 6,441,163, 7,368,565, WO02/16368and WO04/1033272, all of which are expressly incorporated by referencein their entirety.

ADCs containing maytansinoids, methods of making same, and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020;5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described ADCscomprising a maytansinoid designated DM1 linked to the monoclonalantibody C242 directed against human colorectal cancer. The conjugatewas found to be highly cytotoxic towards cultured colon cancer cells,and showed antitumor activity in an in vivo tumor growth assay.

Chari et al., Cancer Research 52:127-131 (1992) describe ADCs in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×105 HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansinoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Auristatins and Dolastatins

In some embodiments, the ADC comprises an anti-CD38 antibody conjugatedto dolastatins or dolostatin peptidic analogs and derivatives, theauristatins (U.S. Pat. Nos. 5,635,483; 5,780,588). Dolastatins andauristatins have been shown to interfere with microtubule dynamics, GTPhydrolysis, and nuclear and cellular division (Woyke et al (2001)Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer(U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998)Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin orauristatin drug moiety may be attached to the antibody through the N(amino) terminus or the C (carboxyl) terminus of the peptidic drugmoiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in “Senter etal, Proceedings of the American Association for Cancer Research, Volume45, Abstract Number 623, presented Mar. 28, 2004 and described in UnitedStates Patent Publication No. 2005/0238648, the disclosure of which isexpressly incorporated by reference in its entirety.

An exemplary auristatin embodiment is MMAE (shown in FIG. 10 wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody drug conjugate; see U.S. Pat. No. 6,884,869 expresslyincorporated by reference in its entirety).

Another exemplary auristatin embodiment is MMAF, shown in FIG. 10wherein the wavy line indicates the covalent attachment to a linker (L)of an antibody drug conjugate (US 2005/0238649, U.S. Pat. Nos. 5,767,237and 6,124,431, expressly incorporated by reference in their entirety):

Additional exemplary embodiments comprising MMAE or MMAF and variouslinker components (described further herein) have the followingstructures and abbreviations (wherein Ab means antibody and p is 1 toabout 8):

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Lubke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. Nos. 5,635,483;5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettitet al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al.Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. PerkinTrans. 1 5:859-863; and Doronina (2003) Nat Biotechnol 21(7):778-784.

Calicheamicin

In other embodiments, the ADC comprises an antibody of the inventionconjugated to one or more calicheamicin molecules. For example, Mylotargis the first commercial ADC drug and utilizes calicheamicin γ1 as thepayload (see U.S. Pat. No. 4,970,198, incorporated by reference in itsentirety). Additional calicheamicin derivatives are described in U.S.Pat. Nos. 5,264,586, 5,384,412, 5,550,246, 5,739,116, 5,773,001,5,767,285 and 5,877,296, all expressly incorporated by reference. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ1I, α2I, α2I, N-acetyl-γ1I, PSAG and θI1 (Hinman et al.,Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research58:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Duocarmycins

CC-1065 (see U.S. Pat. No. 4,169,888, incorporated by reference) andduocarmycins are members of a family of antitumor antibiotics utilizedin ADCs. These antibiotics appear to work through sequence-selectivelyalkylating DNA at the N3 of adenine in the minor groove, which initiatesa cascade of events that result in apoptosis.

Important members of the duocarmycins include duocarmycin A (U.S. Pat.No. 4,923,990, incorporated by reference) and duocarmycin SA (U.S. Pat.No. 5,101,038, incorporated by reference), and a large number ofanalogues as described in U.S. Pat. Nos. 7,517,903, 7,691,962,5,101,038; 5,641,780; 5,187,186; 5,070,092; 5,070,092; 5,641,780;5,101,038; 5,084,468, 5,475,092, 5,585,499, 5,846,545, WO2007/089149,WO2009/017394A1, U.S. Pat. Nos. 5,703,080, 6,989,452, 7,087,600,7,129,261, 7,498,302, and 7,507,420, all of which are expresslyincorporated by reference.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an ADC formed between anantibody and a compound with nucleolytic activity (e.g., a ribonucleaseor a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 andradioactive isotopes of Lu.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc99m or I123, Re186, Re188 and In111 can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate Iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

For compositions comprising a plurality of antibodies, the drug loadingis represented by p, the average number of drug molecules per Antibody.Drug loading may range from 1 to 20 drugs (D) per Antibody. The averagenumber of drugs per antibody in preparation of conjugation reactions maybe characterized by conventional means such as mass spectroscopy, ELISAassay, and HPLC. The quantitative distribution ofAntibody-Drug-Conjugates in terms of p may also be determined.

In some instances, separation, purification, and characterization ofhomogeneous Antibody-Drug-conjugates where p is a certain value fromAntibody-Drug-Conjugates with other drug loadings may be achieved bymeans such as reverse phase HPLC or electrophoresis. In exemplaryembodiments, p is 2, 3, 4, 5, 6, 7, or 8 or a fraction thereof.

The generation of Antibody-drug conjugate compounds can be accomplishedby any technique known to the skilled artisan. Briefly, theAntibody-drug conjugate compounds can include an anti-CD38 antibody asthe Antibody unit, a drug, and optionally a linker that joins the drugand the binding agent.

A number of different reactions are available for covalent attachment ofdrugs and/or linkers to binding agents. This is can be accomplished byreaction of the amino acid residues of the binding agent, for example,antibody molecule, including the amine groups of lysine, the freecarboxylic acid groups of glutamic and aspartic acid, the sulfhydrylgroups of cysteine and the various moieties of the aromatic amino acids.A commonly used non-specific methods of covalent attachment is thecarbodiimide reaction to link a carboxy (or amino) group of a compoundto amino (or carboxy) groups of the antibody. Additionally, bifunctionalagents such as dialdehydes or imidoesters have been used to link theamino group of a compound to amino groups of an antibody molecule.

Also available for attachment of drugs to binding agents is the Schiffbase reaction. This method involves the periodate oxidation of a drugthat contains glycol or hydroxy groups, thus forming an aldehyde whichis then reacted with the binding agent. Attachment occurs via formationof a Schiff base with amino groups of the binding agent. Isothiocyanatescan also be used as coupling agents for covalently attaching drugs tobinding agents. Other techniques are known to the skilled artisan andwithin the scope of the present invention.

In some embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. In otherembodiments, reactive groups are used on the drug and/or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with ananti-CD38 antibody of the invention under appropriate conditions.

It will be understood that chemical modifications may also be made tothe desired compound in order to make reactions of that compound moreconvenient for purposes of preparing conjugates of the invention. Forexample a functional group e.g. amine, hydroxyl, or sulfhydryl, may beappended to the drug at a position which has minimal or an acceptableeffect on the activity or other properties of the drug

Linker Units

Typically, the antibody-drug conjugate compounds comprise a Linker unitbetween the drug unit and the antibody unit. In some embodiments, thelinker is cleavable under intracellular or extracellular conditions,such that cleavage of the linker releases the drug unit from theantibody in the appropriate environment. For example, solid tumors thatsecrete certain proteases may serve as the target of the cleavablelinker; in other embodiments, it is the intracellular proteases that areutilized. In yet other embodiments, the linker unit is not cleavable andthe drug is released, for example, by antibody degradation in lysosomes.

In some embodiments, the linker is cleavable by a cleaving agent that ispresent in the intracellular environment (for example, within a lysosomeor endosome or caveolea). The linker can be, for example, a peptidyllinker that is cleaved by an intracellular peptidase or protease enzyme,including, but not limited to, a lysosomal or endosomal protease. Insome embodiments, the peptidyl linker is at least two amino acids longor at least three amino acids long or more.

Cleaving agents can include, without limitation, cathepsins B and D andplasmin, all of which are known to hydrolyze dipeptide drug derivativesresulting in the release of active drug inside target cells (see, e.g.,Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Peptidyllinkers that are cleavable by enzymes that are present inCD38-expressing cells. For example, a peptidyl linker that is cleavableby the thiol-dependent protease cathepsin-B, which is highly expressedin cancerous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Glylinker (SEQ ID NO: X)). Other examples of such linkers are described,e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference inits entirety and for all purposes.

In some embodiments, the peptidyl linker cleavable by an intracellularprotease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat.No. 6,214,345, which describes the synthesis of doxorubicin with theval-cit linker).

In other embodiments, the cleavable linker is pH-sensitive, that is,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (for example,a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) may be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem.264:14653-14661.) Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond (see, e.g.,U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducingconditions (for example, a disulfide linker). A variety of disulfidelinkers are known in the art, including, for example, those that can beformed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-,SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

In other embodiments, the linker is a malonate linker (Johnson et al.,1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau etal., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3′-N-amide analog(Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released by antibody degradation. (See U.S. Publication No.2005/0238649 incorporated by reference herein in its entirety and forall purposes).

In many embodiments, the linker is self-immolative. As used herein, theterm “self-immolative Spacer” refers to a bifunctional chemical moietythat is capable of covalently linking together two spaced chemicalmoieties into a stable tripartite molecule. It will spontaneouslyseparate from the second chemical moiety if its bond to the first moietyis cleaved. See for example, WO 2007059404A2, WO06110476A2,WO05112919A2, WO2010/062171, WO09/017394, WO07/089149, WO 07/018431,WO04/043493 and WO02/083180, which are directed to drug-cleavablesubstrate conjugates where the drug and cleavable substrate areoptionally linked through a self-immolative linker and which are allexpressly incorporated by reference.

Often the linker is not substantially sensitive to the extracellularenvironment. As used herein, “not substantially sensitive to theextracellular environment,” in the context of a linker, means that nomore than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of thelinkers, in a sample of antibody-drug conjugate compound, are cleavedwhen the antibody-drug conjugate compound presents in an extracellularenvironment (for example, in plasma).

Whether a linker is not substantially sensitive to the extracellularenvironment can be determined, for example, by incubating with plasmathe antibody-drug conjugate compound for a predetermined time period(for example, 2, 4, 8, 16, or 24 hours) and then quantitating the amountof free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotescellular internalization. In certain embodiments, the linker promotescellular internalization when conjugated to the therapeutic agent (thatis, in the milieu of the linker-therapeutic agent moiety of theantibody-drug conjugate compound as described herein). In yet otherembodiments, the linker promotes cellular internalization whenconjugated to both the auristatin compound and the anti-CD38 antibodiesof the invention.

A variety of exemplary linkers that can be used with the presentcompositions and methods are described in WO 2004-010957, U.S.Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S.Publication No. 2006/0024317 (each of which is incorporated by referenceherein in its entirety and for all purposes).

Drug Loading

Drug loading is represented by p and is the average number of Drugmoieties per antibody in a molecule. Drug loading (“p”) may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moremoieties (D) per antibody, although frequently the average number is afraction or a decimal. Generally, drug loading of from 1 to 4 isfrequently useful, and from 1 to 2 is also useful. ADCs of the inventioninclude collections of antibodies conjugated with a range of drugmoieties, from 1 to 20. The average number of drug moieties per antibodyin preparations of ADC from conjugation reactions may be characterizedby conventional means such as mass spectroscopy and, ELISA assay.

The quantitative distribution of ADC in terms of p may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous ADC where p is a certain value from ADCwith other drug loadings may be achieved by means such aselectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8; from about 2 to about 6; from about 3 to about 5; from about3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8;from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shownthat for certain ADCs, the optimal ratio of drug moieties per antibodymay be less than 8, and may be about 2 to about 5. See US 2005-0238649A1 (herein incorporated by reference in its entirety).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number and/or position of linker-drug attachments (suchas thioMab or thioFab prepared as disclosed herein and in WO2006/034488(herein incorporated by reference in its entirety)).

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography.

In some embodiments, a homogeneous ADC with a single loading value maybe isolated from the conjugation mixture by electrophoresis orchromatography.

Methods of Determining Cytotoxic Effect of ADCs

Methods of determining whether a Drug or Antibody-Drug conjugate exertsa cytostatic and/or cytotoxic effect on a cell are known. Generally, thecytotoxic or cytostatic activity of an Antibody Drug conjugate can bemeasured by: exposing mammalian cells expressing a target protein of theAntibody Drug conjugate in a cell culture medium; culturing the cellsfor a period from about 6 hours to about 5 days; and measuring cellviability. Cell-based in vitro assays can be used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of the Antibody Drug conjugate.

For determining whether an Antibody Drug conjugate exerts a cytostaticeffect, a thymidine incorporation assay may be used. For example, cancercells expressing a target antigen at a density of 5,000 cells/well of a96-well plated can be cultured for a 72-hour period and exposed to 0.5μCi of ³H-thymidine during the final 8 hours of the 72-hour period. Theincorporation of ³H-thymidine into cells of the culture is measured inthe presence and absence of the Antibody Drug conjugate.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) can be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane; swelling of the cell, and ruptureof the plasma membrane. Apoptosis is typically characterized by membraneblebbing, condensation of cytoplasm, and the activation of endogenousendonucleases. Determination of any of these effects on cancer cellsindicates that an Antibody Drug conjugate is useful in the treatment ofcancers.

Cell viability can be measured by determining in a cell the uptake of adye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Pageet al., 1993, Intl. J. Oncology 3:473-476). In such an assay, the cellsare incubated in media containing the dye, the cells are washed, and theremaining dye, reflecting cellular uptake of the dye, is measuredspectrophotometrically. The protein-binding dye sulforhodamine B (SRB)can also be used to measure cytoxicity (Skehan et al., 1990, J. Natl.Cancer Inst. 82:1107-12).

Alternatively, a tetrazolium salt, such as MTT, is used in aquantitative colorimetric assay for mammalian cell survival andproliferation by detecting living, but not dead, cells (see, e.g.,Mosmann, 1983, J. Immunol. Methods 65:55-63).

Apoptosis can be quantitated by measuring, for example, DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has beendescribed by Duke and Cohen, Current Protocols in Immunology (Coligan etal. eds., 1992, pp. 3.17.1-3.17.16). Cells also can be labeled with aDNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide)and the cells observed for chromatin condensation and margination alongthe inner nuclear membrane. Other morphological changes that can bemeasured to determine apoptosis include, e.g., cytoplasmic condensation,increased membrane blebbing, and cellular shrinkage.

The presence of apoptotic cells can be measured in both the attached and“floating” compartments of the cultures. For example, both compartmentscan be collected by removing the supernatant, trypsinizing the attachedcells, combining the preparations following a centrifugation wash step(e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., bymeasuring DNA fragmentation). (See, e.g., Piazza et al., 1995, CancerResearch 55:3110-16).

In vivo, the effect of a therapeutic composition of the anti-CD38antibody of the invention can be evaluated in a suitable animal model.For example, xenogeneic cancer models can be used, wherein cancerexplants or passaged xenograft tissues are introduced into immunecompromised animals, such as nude or SCID mice (Klein et al., 1997,Nature Medicine 3: 402-408). Efficacy can be measured using assays thatmeasure inhibition of tumor formation, tumor regression or metastasis,and the like.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

Antibody Compositions for In Vivo Administration

Formulations of the antibodies used in accordance with the presentinvention are prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to provide antibodies with otherspecificities. Alternatively, or in addition, the composition maycomprise a cytotoxic agent, cytokine, growth inhibitory agent and/orsmall molecule antagonist. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration should besterile, or nearly so. This is readily accomplished by filtrationthrough sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

When encapsulated antibodies remain in the body for a long time, theymay denature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Administrative Modalities

The antibodies and chemotherapeutic agents of the invention areadministered to a subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the antibody is preferred.

Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Thus for B cell tumors, the subject may experience a decrease in theso-called B symptoms, i.e., night sweats, fever, weight loss, and/orurticaria. For pre-malignant conditions, therapy with an anti-CD38therapeutic agent may block and/or prolong the time before developmentof a related malignant condition, for example, development of multiplemyeloma in subjects suffering from monoclonal gammopathy of undeterminedsignificance (MGUS).

An improvement in the disease may be characterized as a completeresponse. By “complete response” is intended an absence of clinicallydetectable disease with normalization of any previously abnormalradiographic studies, bone marrow, and cerebrospinal fluid (CSF) orabnormal monoclonal protein in the case of myeloma.

Such a response may persist for at least 4 to 8 weeks, or sometimes 6 to8 weeks, following treatment according to the methods of the invention.Alternatively, an improvement in the disease may be categorized as beinga partial response. By “partial response” is intended at least about a50% decrease in all measurable tumor burden (i.e., the number ofmalignant cells present in the subject, or the measured bulk of tumormasses or the quantity of abnormal monoclonal protein) in the absence ofnew lesions, which may persist for 4 to 8 weeks, or 6 to 8 weeks.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the anti-CD38antibodies used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art.

An exemplary, non-limiting range for a therapeutically effective amountof an anti-CD38 antibody used in the present invention is about 0.1-100mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, suchas about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about1, or about 3 mg/kg. In another embodiment, the antibody is administeredin a dose of 1 mg/kg or more, such as a dose of from 1 to 20 mg/kg, e.g.a dose of from 5 to 20 mg/kg, e.g. a dose of 8 mg/kg.

A medical professional having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, a physician or a veterinarian couldstart doses of the medicament employed in the pharmaceutical compositionat levels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved.

In one embodiment, the anti-CD38 antibody is administered by infusion ina weekly dosage of from 10 to 500 mg/kg such as of from 200 to 400 mg/kgSuch administration may be repeated, e.g., 1 to 8 times, such as 3 to 5times. The administration may be performed by continuous infusion over aperiod of from 2 to 24 hours, such as of from 2 to 12 hours.

In one embodiment, the anti-CD38 antibody is administered by slowcontinuous infusion over a long period, such as more than 24 hours, ifrequired to reduce side effects including toxicity.

In one embodiment the anti-CD38 antibody is administered in a weeklydosage of from 250 mg to 2000 mg, such as for example 300 mg, 500 mg,700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4to 6 times. The administration may be performed by continuous infusionover a period of from 2 to 24 hours, such as of from 2 to 12 hours. Suchregimen may be repeated one or more times as necessary, for example,after 6 months or 12 months. The dosage may be determined or adjusted bymeasuring the amount of compound of the present invention in the bloodupon administration by for instance taking out a biological sample andusing anti-idiotypic antibodies which target the antigen binding regionof the anti-CD38 antibody.

In a further embodiment, the anti-CD38 antibody is administered onceweekly for 2 to 12 weeks, such as for 3 to 10 weeks, such as for 4 to 8weeks.

In one embodiment, the anti-CD38 antibody is administered by maintenancetherapy, such as, e.g., once a week for a period of 6 months or more.

In one embodiment, the anti-CD38 antibody is administered by a regimenincluding one infusion of an anti-CD38 antibody followed by an infusionof an anti-CD38 antibody conjugated to a radioisotope. The regimen maybe repeated, e.g., 7 to 9 days later.

As non-limiting examples, treatment according to the present inventionmay be provided as a daily dosage of an antibody in an amount of about0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on atleast one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 afterinitiation of treatment, or any combination thereof, using single ordivided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combinationthereof.

In some embodiments the anti-CD38 antibody molecule thereof is used incombination with one or more additional therapeutic agents, e.g. achemotherapeutic agent. Non-limiting examples of DNA damagingchemotherapeutic agents include topoisomerase I inhibitors (e.g.,irinotecan, topotecan, camptothecin and analogs or metabolites thereof,and doxorubicin); topoisomerase II inhibitors (e.g., etoposide,teniposide, and daunorubicin); alkylating agents (e.g., melphalan,chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine,semustine, streptozocin, decarbazine, methotrexate, mitomycin C, andcyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, andcarboplatin); DNA intercalators and free radical generators such asbleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitibine,gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine,pentostatin, and hydroxyurea).

Chemotherapeutic agents that disrupt cell replication include:paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, andrelated analogs; thalidomide, lenalidomide, and related analogs (e.g.,CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinibmesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-κBinhibitors, including inhibitors of IκB kinase; antibodies which bind toproteins overexpressed in cancers and thereby downregulate cellreplication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab);and other inhibitors of proteins or enzymes known to be upregulated,over-expressed or activated in cancers, the inhibition of whichdownregulates cell replication.

In some embodiments, the antibodies of the invention can be used priorto, concurrent with, or after treatment with Velcade® (bortezomib).

Diagnostic Uses

The anti-CD38 antibodies provided also find use in the in vitro or invivo imaging of tumors or other disease states associated with CD38. Insome embodiments, the antibodies described herein are used for bothdiagnosis and treatment, or for diagnosis alone. When anti-CD38antibodies are used for both diagnosis and treatment, some embodimentsrely on two different anti-CD38 antibodies to two different epitopes,such that the diagnostic antibody does not compete for binding with thetherapeutic antibody, although in some cases the same antibody can beused for both. For example, in some instances, the Ab19 antibody is useddiagnostically (generally labeled as discussed below) while Ab79 is usedtherapeutically, or vice versa. Thus included in the invention arecompositions comprising a diagnostic antibody and a therapeuticantibody, and in some embodiments, the diagnostic antibody is labeled asdescribed herein. In addition, the composition of therapeutic anddiagnostic antibodies can also be co-administered with other drugs asoutlined herein.

In many embodiments, a diagnostic antibody is labeled. By “labeled”herein is meant that the antibodies disclosed herein have one or moreelements, isotopes, or chemical compounds attached to enable thedetection in a screen or diagnostic procedure. In general, labels fallinto several classes: a) immune labels, which may be an epitopeincorporated as a fusion partner that is recognized by an antibody, b)isotopic labels, which may be radioactive or heavy isotopes, c) smallmolecule labels, which may include fluorescent and colorimetric dyes, ormolecules such as biotin that enable other labeling methods, and d)labels such as particles (including bubbles for ultrasound labeling) orparamagnetic labels that allow body imagining. Labels may beincorporated into the antibodies at any position and may be incorporatedin vitro or in vivo during protein expression, as is known in the art.

Diagnosis can be done either in vivo, by administration of a diagnosticantibody that allows whole body imaging as described below, or in vitro,on samples removed from a patient. “Sample” in this context includes anynumber of things, including, but not limited to, bodily fluids(including, but not limited to, blood, urine, serum, lymph, saliva, analand vaginal secretions, perspiration and semen), as well as tissuesamples such as result from biopsies of relevant tissues.

In some embodiments, in vivo imaging is done, including but not limitedto ultrasound, CT scans, X-rays, MRI and PET scans, as well as opticaltechniques, such as those using optical labels for tumors near thesurface of the body.

In vivo imaging of tumors associated with CD38 may be performed by anysuitable technique. For example, ⁹⁹Tc-labeling or labeling with anotherβ-ray emitting isotope may be used to label anti-CD38 antibodies.Variations on this technique may include the use of magnetic resonanceimaging (Mill) to improve imaging over gamma camera techniques. Similarimmunoscintigraphy methods and principles are described in, e.g.,Srivastava (ed.), Radiolabeled Monoclonal Antibodies For Imaging AndTherapy (Plenum Press 1988), Chase, “Medical Applications ofRadioisotopes,” in Remington's Pharmaceutical Sciences, 18th Edition,Gennaro et al., (eds.), pp. 624-652 (Mack Publishing Co., 1990), andBrown, “Clinical Use of Monoclonal Antibodies,” in Biotechnology AndPharmacy 227-49, Pezzuto et al., (eds.) (Chapman & Hall 1993).

In one embodiment, the present invention provides an in vivo imagingmethod wherein an anti-CD38 antibody is conjugated to adetection-promoting agent, the conjugated antibody is administered to ahost, such as by injection into the bloodstream, and the presence andlocation of the labeled antibody in the host is assayed. Through thistechnique and any other diagnostic method provided herein, the presentinvention provides a method for screening for the presence ofdisease-related cells in a human patient or a biological sample takenfrom a human patient.

For diagnostic imaging, radioisotopes may be bound to an anti-CD38antibody either directly, or indirectly by using an intermediaryfunctional group. Useful intermediary functional groups includechelators, such as ethylenediaminetetraacetic acid anddiethylenetriaminepentaacetic acid (see for instance U.S. Pat. No.5,057,313). In such diagnostic assays involving radioisotope-conjugatedanti-CD38 antibodies, the dosage of conjugated anti-CD38 antibodydelivered to the patient typically is maintained at as low a level aspossible through the choice of isotope for the best combination ofminimum half-life, minimum retention in the body, and minimum quantityof isotope, which will permit detection and accurate measurement.

In addition to radioisotopes and radio-opaque agents, diagnostic methodsmay be performed using anti-CD38 antibodies that are conjugated to dyes(such as with the biotin-streptavidin complex), contrast agents,fluorescent compounds or molecules and enhancing agents (e.g.paramagnetic ions) for magnetic resonance imaging (MRI) (see, e.g., U.S.Pat. No. 6,331,175, which describes MRI techniques and the preparationof antibodies conjugated to a MRI enhancing agent). Suchdiagnostic/detection agents may be selected from agents for use inmagnetic resonance imaging, and fluorescent compounds.

In order to load an anti-CD38 antibody with radioactive metals orparamagnetic ions, it may be necessary to react it with a reagent havinga long tail to which are attached a multiplicity of chelating groups forbinding the ions. Such a tail may be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chain havingpendant groups to which can be bound chelating groups such as, e.g.,porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes,and like groups known to be useful for this purpose.

Chelates may be coupled to anti-CD38 antibodies using standardchemistries. A chelate is normally linked to an anti-CD38 antibody by agroup that enables formation of a bond to the molecule with minimal lossof immunoreactivity and minimal aggregation and/or internalcross-linking.

Examples of potentially useful metal-chelate combinations include2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used withdiagnostic isotopes in the general energy range of 60 to 4,000 keV, suchas ¹²⁵I, ¹²³I, ¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁹⁹Tc, ⁹⁴Tc, ¹¹C, ¹³N,⁵O, and ⁷⁶Br, for radio-imaging.

Labels include a radionuclide, a radiological contrast agent, aparamagnetic ion, a metal, a fluorescent label, a chemiluminescentlabel, an ultrasound contrast agent and a photoactive agent. Suchdiagnostic agents are well known and any such known diagnostic agent maybe used. Non-limiting examples of diagnostic agents may include aradionuclide such as ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ⁹⁴mTc, ⁹⁴Tc, ⁹⁹mTc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I,¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P, ¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ⁵²mMn, ⁵⁵Co,⁷²As, ⁷⁵Br, ⁷⁶Br, ⁸²mRb, ⁸³Sr, or other γ-, β-, or positron-emitters.

Paramagnetic ions of use may include chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III). Metalcontrast agents may include lanthanum (III), gold (III), lead (II) orbismuth (III).

Ultrasound contrast agents may comprise liposomes, such as gas filledliposomes. Radiopaque diagnostic agents may be selected from compounds,barium compounds, gallium compounds, and thallium compounds.

These and similar chelates, when complexed with non-radioactive metals,such as manganese, iron, and gadolinium may be useful for MRI diagnosticmethods in connection with anti-CD38 antibodies. Macrocyclic chelatessuch as NOTA, DOTA, and TETA are of use with a variety of metals andradiometals, most particularly with radionuclides of gallium, yttrium,and copper, respectively. Such metal-chelate complexes may be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelates such as macrocyclic polyethers, which are of interestfor stably binding nuclides, such as ²²³Ra may also be suitable indiagnostic methods.

Thus, the present invention provides diagnostic anti-CD38 antibodyconjugates, wherein the anti-CD38 antibody conjugate is conjugated to acontrast agent (such as for magnetic resonance imaging, computedtomography, or ultrasound contrast-enhancing agent) or a radionuclidethat may be, for example, a γ-, β-, α-, Auger electron-, orpositron-emitting isotope.

Anti-CD38 antibodies may also be useful in, for example, detectingexpression of an antigen of interest in specific cells, tissues, orserum. For diagnostic applications, the antibody typically will belabeled with a detectable moiety for in vitro assays. As will beappreciated by those in the art, there are a wide variety of suitablelabels for use in in vitro testing. Suitable dyes for use in this aspectof the invention include, but are not limited to, fluorescent lanthanidecomplexes, including those of Europium and Terbium, fluorescein,rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin,methyl-coumarins, quantum dots (also referred to as “nanocrystals”; seeU.S. Ser. No. 09/315,584, hereby incorporated by reference), pyrene,Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, Cydyes (Cy3, Cy5, etc.), alexa dyes (including Alexa, phycoerythin,bodipy, and others described in the 6th Edition of the Molecular ProbesHandbook by Richard P. Haugland, hereby expressly incorporated byreference.

Stained tissues may then be assessed for radioactivity counting as anindicator of the amount of CD38-associated peptides in the tumor. Theimages obtained by the use of such techniques may be used to assessbiodistribution of CD38 in a patient, mammal, or tissue, for example inthe context of using CD38 as a biomarker for the presence of invasivecancer cells.

Articles of Manufacture

In other embodiments, an article of manufacture containing materialsuseful for the treatment of the disorders described above is provided.The article of manufacture comprises a container and a label. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. The containers may be formed from a variety of materials such asglass or plastic. The container holds a composition which is effectivefor treating the condition and may have a sterile access port (forexample the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). Theactive agent in the composition is the antibody. The label on, orassociated with, the container indicates that the composition is usedfor treating the condition of choice. The article of manufacture mayfurther comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

EXAMPLES

The following examples are offered to illustrate, but not to limit theinvention.

Example 1: Construction of Expression Vectors Comprising PolynucleotidesEncoding Human, Cynomolgus Monkey, and Mouse CD38

To construct a vector expressing human CD38 (huCD38), a polynucleotideencoding huCD38 was isolated from cDNA obtained from OrigeneTechnologies Trueclone® human. The isolated huCD38 was cloned into astable expression vector (XOMA, Inc.) containing the neomycin resistance(nee) gene, which allowed for the selection of G418(Geneticin)-resistant transfectants. The huCD38 gene present in theselected transfectants was sequenced to identify any sequence errors.Errors in the sequence that deviated from Genbank accession NM_001775were corrected by PCR site-directed mutagenesis. The final vector DNAwas confirmed by 5′ sequencing.

To construct a vector expressing cynomolgus monkey CD38 (cyCD38), apolynucleotide encoding cyCD38 was isolated from DNA obtained fromBiochain Institute's cDNA-monkey (cynomolgus)-normal spleen tissue. Theisolated cyCD38 was cloned into a stable expression vector (XOMA, Inc.)containing the nee gene, which allowed for the selection of G418(Geneticin)-resistant transfectants. The cyCD38 gene present in theselected transfectants was sequenced to identify any sequence errors.Errors in the sequence that deviated from Genbank accession AY555148were corrected by PCR sitedirected mutagenesis. The final vector DNA wasconfirmed by sequencing.

To construct a vector expressing mouse CD38 (moCD38), a polynucleotideencoding moCD38 was isolated from DNA obtained from Origene's TrueORFcollection. The isolated moCD38 was cloned into a stable expressionvector (XOMA, Inc.) containing the neo^(R) gene, which allowed for theselection of G418 (Geneticin)-resistant transfectants. The moCD38 genepresent in the selected transfectants was sequenced to identify anysequence errors. Errors in the sequence that deviated from Genbankaccession NM_007646 were corrected by PCR site-directed mutagenesis. Thefinal vector DNA was confirmed by sequencing.

Example 2: Development of CD38 Expressing Chinese Hamster Ovary (CHO)Cells

For development of CHO cells expressing huCD38, muCD38 and cyCD38, CHOcells were transfected with linearized DNA. After one week underselection, the cells were sorted by flow cytometry and the highesthuCD38, muCD38 or cyCD38 expressing cells (top 15%) were plated in96-well plates to generate single colonies. The remaining cells werealso plated under selection to generate backup colonies. Approximately12-14 days after plating, single colonies were identified andtransferred to 96-deep-well plates. Clones were screened by FACSanalysis after the second passage. Top producing clones were passagedand expanded to shake flasks. The top 2 clones were frozen and/orcultured for mycoplasmal AVA testing and scale-up.

To construct a luciferase reporter for disseminated xenograft models, acommercial vector containing the CMV promoter/luciferase gene/neomycinselectable marker (Promega, Madison, Wis.) was used to generate stabletransfectant line in Daudi Burkitt's lymphoma cells.

Example 3: Phage Display Libraries and Screening of Agents that BindCD38

Selection of target specific antibody from a phage display library wascarried out according to methods described by Marks et al. (2004,Methods Mol. Biol. 248:161-76). Briefly, the phage display library wasincubated with 100 pmols of biotinylated CD38 at room temperature for 1hr and the complex formed was then captured using 100 μL of Streptavidinbead suspension (DYNABEADS® M-280 Streptavidin, Invitrogen).Non-specific phages were removed by washing the beads with wash buffer(5% milk in PBS). Bound phages were eluted with 0.5 ml of 100 nMtriethyleamine (TEA) and immediately neutralized by addition of an equalvolume of 1M TRIS-CI, pH 7.4. The eluted phage pool was used to infectTG1 E. coli cells growing in logarithmic phase and phagemid was rescuedas described in Marks et al., Id. Selection was repeated for a total ofthree rounds.

Alternatively, phage display libraries were panned against immobilizedCD38 (R&D systems) to identify a panel of antibody fragments with theability to bind CD38. Panning was carried out using standard protocols(see, e.g., Methods in Molecular Biology, vol. 178: Antibody PhageDisplay: Methods and Protocols Edited by: P. M. O'Brien and R. Aitken,Humana Press; “Panning of Antibody Phage-Display Libraries,” Coomber, D.W. J., pp. 133-145, and “Selection of Antibodies Against BiotinylatedAntigens,” Chames et al., pp. 147-157). Briefly, three wells of a NUNC®MAXISORP plate were coated with 50 μL of recombinant CD38 (R&D Systems)at a concentration of 10 μg/ml in PBS. After overnight incubation at 4°C., free binding sites were blocked with 5% milk in PBS for one hour atroom temperature. Approximately 200 μL of phage library in 5% milk/PBSwas then added to the blocked wells and incubated at room temperaturefor approximately one to two hours. Wells were washed and bound phagewas eluted using standard methods (see, e.g., Sam brook and Russell,Molecule Cloning: A Laboratory Manual, 3^(rd) Edition, Cold SpringHarbor Laboratory Press, 2001). Eluted phage was amplified via infectingE. coli TG 1 host cells in logarithmic growth phase. Infected TG 1 cellswere recovered by centrifugation at 2,500 RPM for five minutes, platedonto 15 cm 2YT-ampicillin-2% glucose agar plates, and incubated at 30°C. overnight. The panning process was then repeated using the amplifiedphage. The cycle of panning, elution, and amplification was repeated forthree rounds.

After panning completion, single colonies from the plated TG1 cells wereused to inoculate media in 96-well plates. Microcultures were grown toan OD600 of 0.6, at which point expression of soluble scFv was inducedby addition of 1 mM IPTG and overnight incubation in a shaker at 30° C.Bacteria were pelleted by centrifugation and periplasmic extract wasused to test scFv binding to immobilized CD38 using a standard ELISAassay and a FACS-binding assay.

For FACS binding screen, CHO cells stably expressing CD38 were used toscreen scFvs in periplasmic extract (PPE) for their ability to bindnative, membrane bound CD38. Parental and CHO transfectants (Human CD38or Cyno CD38 or Mouse CD38-expressing cell lines) were resuspendedseparately at 2×10⁶ cells/ml in PBS (Life Technologies), 0.5% BSA(SigmaAldrich), and 0, 1 NaN3 (Sigma-Aldrich) (FACS buffer). ParentalCHO cells not expressing CD38 were used as a negative control. Twentyfive μL aliquots of the cells were plated in Vbottomed 96-well plates(Costar Cat #3897) and 25 μL of periplasmic extract containingmyc-tagged scFv antibody fragment was added to the cells, then themixture was incubated at 4° C. for 30 minutes. The cells were thenwashed twice after which the pellet was resuspended in 25 μL of mouseanti c-myc (1/1000 in FACS buffer)(Roche) and again incubated at 4° C.for 30 minutes. The cells were then washed twice and resuspended in 25μL of 1/200 dilution anti-mouse IgG-PE in FACS buffer (Jackson labs) andagain incubated at 4° C. for 30 minutes. The cells were then washedtwice to remove excess unbound antibody and resuspended in 70 μL FACSbuffer and analysed on a BD FACScan®. The acquired data was evaluatedusing FlowJo software (TreeStar, Inc.). Positive samples were identifiedby comparing the median fluorescence intensity of the CD38 transfectedCHO cell relative to the median fluorescence intensity of the parentalCHO cell-line (CD38⁻).

Antibody clones that bound human CD38 were sequenced to identify uniqueclones. The unique scFV clones were then ranked based on off-ratesdetermined by Biacore® analysis. 200RU to 500RU of human recombinantCD38 (R&D Systems' cat #2404-AC or equivalent) were immobilized bystandard amine coupling chemistry (Biacore®) to a CM5 or equivalentchip. A reference spot was also prepared which was activated and thenblocked without the immobilization of the protein. This was done bydiluting the antigen to 1-3 μg/ml in acetate buffer, pH 5.0, andinjecting over the activated surface until required level wasimmobilized (3-5) minutes. The surface was then blocked withethanolamine. Periplasmic extracts were diluted one-to-one with theassay running buffer 10 mM HEPES, 150 mM NaCl, 3 mM EDTA(ethylenediaminetetraacetic acid), and 0.05% polysorbate 20 at pH 7.4with 2 mg/mL BSA (bovine serum albumin)). The diluted periplasmicextract was injected over the surface plasmon resonance (SPR) surfacesat 30 minute for 300 seconds with an additional 900 seconds ofdissociation time monitored. Regeneration was with a single 8-secondinjection of 100 mM HCl. Data from the refer˜nce sp,pts were subtractedfrom the data from the active surface, then dissociation curves were fitusing the 1:1 dissociation model in the Biacore® T100 software.

Top-ranking scFV clones were converted to IgG1 antibodies. The FACSbinding screen was repeated on the IgG1 reformatted clones usingparental CHO cells and CHO cells expressing human, murine and cynomolgusCD38 to ensure binding properties were retained and to assess speciescross-reactivity. FACS characterization of IgG-reformatted clones wasconducted as described above, but the steps consisting of the additionof anti-c-myc antibody and anti-mouse IgG-PE were replaced by a singlestep in which binding of full-length human IgG was detected by theaddition of phycoerythrin conjugated anti-human IgG (Jackson Labs).

Example 4: In Vitro Cell-Based Assays of IgG-Reformatted Clones

About 150 clones were reformatted as human IgG1 antibodies and five(Ab19, Ab43, Ab72, Ab79, and Ab110) were fully evaluated using a panelof assays, as described below. Performance of IgG-reformatted clones inboth in vitro and in vivo assays was compared to two antibodies, BMTK4-1(also called benchmark-1, BM-1, or BMTK-1) (SEQ ID NOs:24 and 25; heavyand light chain variable regions) and BMTK4-2 (also called benchmark-2,BM-2, or BMTK-2) (SEQ ID NOs: 26 and 27; heavy and light chain variableregions), the amino acid sequences of which were derived from thesequences of known anti-CD38 antibodies daratumumab (also calledHuMax-CD38, disclosed in International Publication No. WO 06/099875) andSAR650984 (disclosed in International Publication No. WO 08/047242),respectively. Palivizumab (SYNAGIS®) (MedImmune), a clinically approvedantibody that recognizes respiratory syncytial virus served as anegative control for CD38 binding.

Example 5: Detection of Ab79 Binding by Immunofluorescence

Alexa Fluor®-488 dye labeled Ab79 was applied to frozen sections ofnormal human colorectal tissue, prostate, and lymph. Alexa Fluor®-488dye labeled Palivizumab (Synagis®) served as a negative stainingcontrol. The resulting immunofluorescent images are shown in FIG. 4. Thestaining pattern observed for Ab79 was identical to that seen with acommercially available polyclonal anti-CD38 antibody on normal humancolorectal tissue, prostate and lymph node (data not shown).

Alexa Fluor®-488 dye labeled Ab79 was also applied to normal andmultiple myeloma bone marrow specimens (FIG. 5). Whereas Ab79 bound to10% of the cells from normal bone marrow, >90% of the multiple myelomabone marrow cells, in 4 out of 4 tested samples, showed Ab79 binding.

The ability of Ab79 to bind to a number of cell lines (MOLP-8, DAUDI,RPMI, and MCF7) was also examined. MOLP-8 (human multiple myeloma),DAUDI (lymphoblast derived from a patient with Burkitt's lymphoma), andRPMI (cell line established from patient with chronic myelogenesisleukemia) cells all showed binding by Ab79. The breast cancer line,MCF7, appeared largely negative for Ab79 binding (FIG. 6).

The Alexa Fluor® 488-conjugated antibodies were stained on 8 μm cryostatfrozen sections, which were fixed in an ethanol/acetone mixture for 5min followed by incubation with the antibodies for 1 hour at roomtemperature in a humidity-controlled chamber. The sections were thenwashed, a DAPI containing mountant (Vector Laboratories, cat # H1500)was added, and a coverslip was applied.

Example 6: Evaluation of Ab79 Expression on Multiple Myeloma (MM) andChronic Lymphocytic Leukemia (CLL)

Ab79 binding to bone marrow samples from multiple myeloma patients wasanalyzed by flow cytometry either after enrichment for CD138⁺ cells orby gating on CD138⁺CD45^(−/lo) cells (FIG. 7A). Ab79 was found to beexpressed on >95% of cells from four out of six multiple myelomasamples. The binding pattern of Ab79 appeared largely similar to that ofan anti-CD38 antibody used in clinical laboratories. Further, Ab79 boundcells from patients with chronic lymphocytic leukemia (FIG. 7B).

In order to measure Ab79 binding to MM and CLL by FACS patient sampleswere processed within 24 hours. Peripheral blood mononuclear cells wereisolated by Ficoll-Paque™ (GE Healthcare) according to themanufacturer's instructions. Expression analysis was performed using thefollowing panels of antibodies with clones in parentheses. MM panel:Ab79-Alexa Fluor®-488, CD45-PerCP(2D1), CD138-APC (MI15). CLL panel:Ab79-Alexa Fluor®-488; CD5-PE (UCHT2), CD45-PerCP(2D1), CD19-APC(SJ25C1). 5 μL of the PE, PerCP, or APC labeled antibody or 10 μL ofAlexa Fluor®-488 labeled antibody or isotype control was added to eachwell or tube containing either 100 μL of 0.2×106 PBMCs or CD138 enrichedcells from bone marrow aspirate. samples were incubated for 30 mins atroom temperature following which red blood cells were lysed using BDPharmlyse, according to the manufacturer's instructions. All sampleswere washed three times in FACS buffer. Samples were fixed in 1%paraformaldehyde and analyzed on a BD FACSCanto™ II or BD FACSCaliber™.

Example 7: Anti-CD38 Induced CDC Assays

Cynomolgus cross-reactive clones were tested for the ability to inducecomplement-dependent cytotoxicity (CDC). MOLP-8 cells were plated at adensity of 10,000 cells per well in a black 96-well flat-bottom tissueculture plate in 50 μL of complete media (RPMI supplemented with 10%fetal bovine serum). 50 μL of 2× anti-CD38 antibody, control IgGantibody, or media alone was added to each well and left to incubate atroom temperature for 10 min. Varying amounts (2-15 μL) of purifiedrabbit complement (cat # CL 3441 Cedarlane Laboratories, Canada),depending upon the cell line was added to each well except controlwells. After one hour incubation at 37° C., plates were brought to roomtemperature, 100 μL of cell titer CytoTox Glo™ reagent (PromegaG7571/G7573) was added per well, the plate shaken for 5 to 7 min andluminescence read on an EnVision® (Perkin Elmer) luminescence platereader. Conditions tested: cells alone; cells+complement; cells+IgGcontrol+complement; cells+antibody+complement. % CDC was calculatedusing the following equation:

100−(RLU_(T)/RLU_(C))×100),

where RLU_(T) is the relative luminescence units of the test sample andRLU_(C) is the relative luminescence units of the sample with complementalone. Statistical analysis was performed using PRISM software. EC₅₀values, as determined from plots of % CDC versus antibody concentration,are shown in Table 1.

Example 8: Anti-CD38 Induced ADCC Assays

Antibody-dependent cell-mediated cytotoxicity (ADCC) was assessed usingDaudi, MOLP-8, and RPMI-8226 cell lines as target cells. PBMCs wereisolated as effector cells by Ficoll-Plaque™ separation from buffy coator LRS which were obtained from the Stanford Blood Center (Palo Alto,Calif.). Specimens were diluted 1:3 with 2% FBS in PBS. 15 mL ofFicoll-Plaque™ (GE Healthcare) was gently layered under 35 mL of dilutedspecimen and centrifuged at 1800 rpm (brake off) for 25 min. The cloudyinterphase containing PBMCs was collected, washed 3 times in 2% FBS inPBS and frozen aliquots of 50×106 cells/mL per aliquot in 10% DMSO/FBS.Frozen aliquots of PBMCs were thawed and cultured overnight in 10%FBS/RPMI+5 ng/mL recombinant human IL2 (R & D systems #202-IL) at 2×106per mL, when needed.

For the ADCC assay, all steps were performed in complete media. 5000target cells were plated per well in a 96-well plate in 50 μL of 3×anti-CD38, control IgG, or media alone was added followed by 50 μL ofhuman effector PBMCs at a ration of between 1:25 to 1:50 target:effector(T:E) cells. Plates were briefly centrifuged for ˜30 seconds at 800 rpmto bring all cells into close proximity. After 4 hrs at 37° C., plateswere centrifuged at 1100 rpm for 5 min and 100 μL supernatanttransferred to a white plate. 100 μL CytoTox Glo™ reagent (Promega cat #G9292) was added to the supernatant and plates were left to shake for20-30 mins at RT. Luminescence was read on an EnVision® (Perkin Elmer)luminescence plate reader and percent specific lysis was calculatedusing the following equation:

(RLU_(T)/RLU_(E/T))/(RLU_(L)/RLU_(E/T))×100

where RLU_(T) is the relative luminescence units of the test sample andRLU_(E/T) is the relative luminescence units of the sample containingtarget cells and effector cells alone, and RLU_(L) is the relativeluminescence units for cells lysed with Triton X-100. Statisticalanalysis was performed using PRISM software. EC₅₀ values, as determinedfrom plots of % specific lysis versus antibody concentration, are shownin Table 1.

TABLE 1 CDC, ADCC, and Agonist Activity for IgG-Refomatted AntibodiesCDC ADCC ADCC ADCC Apoptosis EC50 nM EC50 nM EC50 nM EC50 nM EC50 nMAntibody (MOLP-8) (DAUDI) (MOLP-8) (RPMI-8226) (DAUDI) BM-1 0.48 ± 0.160.03 ± 0.02 0.036 ± 0.013 0.13 ± 0.03 0.057 BM-2 0.65 ± 0.18 0.04 ± 0.020.024 ± 0.005 0.15 ± 0.04 0.062 Ab19 0.98 ± 0.26 0.08 ± 0.03 0.038 ±0.008 0.46 ± 0.15 0.032 Ab43 2.2 0.12 ± 0.09 0.027 ± 0.018 3.84 ± 1.341.56 Ab72 0.66 ± 0.49 0.14 ± 0.12 0.193 ± 0.037 2.35 ± 0.99 0.35 Ab79 1.1 ± 0.39 0.03 ± 0.02 0.047 ± 0.012 0.46 ± 0.19 0.048 Ab110 1.99 ±0.71 0.24 ± 0.17 0.874 ± 0.804 2.98 ± 0.91 0.40 Ab164 2.00 ± 0.83 ND0.165 ± 0.154  1.2 ± 0.24 0.31

Example 9: Affinity Determination by FACS

MOLP-8 cells expressing CD38 were suspended in 1% FBS buffer at a viablecell concentration of approximately 2 million cells/mL. mAbs to betested were serially diluted (2-fold) across wells over two 96-wellplates in 1×PBS. The last well of each titration contained buffer only.Additional PBS and cell suspensions were added to each well so that thefinal volume was 300 ˜L/well and each well contained approximately100,000 cells. The mAbs are listed below with the corresponding finalmAb binding site concentration (2× molecular concentration) range usedfor the titrations:

Benchmark 1, [mAb]bindingsite=50.8 nM-49.7 pM

Benchmark 2, [mAb]bindingsi,e=49.5 nM-48.3 pM

Ab43, [mAb]binding site=49.3 nM-48.2 pM

Ab110, [mAb]bindingsite=204 nM-49.9 pM

Ab79, [mAb]binding Site=103 nM-25.3 pM

Ab72, [mAb]binding Site=103 nM-25.2 pM

Ab19, [mAb]bindingsite=100 nM-12.2 pM.

The plates were placed into a plate shaker for 5 hours at 4° C., afterwhich the plates were washed 3 times at 4° C. with 1×PBS. 200 μl of 99nM Cy5 goat anti-human IgG Fc specific polyclonal antibody (JacksonImmunoResearch Laboratories, #109-175-008) was then added to each well,and the plates were shaken for 30 minutes at 4° C. The plates were againwashed 2× at 4° C. with 1×PBS, then a FACSCanto™ II HTS flow cytometerwas used to record the mean fluorescence intensity (MFI) of 5000 eventsfor each well containing a unique mAb binding site concentration. A plotof the Mean Fluorescence Intensity as a function of the antibody bindingsite concentration was fit nonlinearly with Scientist 3.0 software usingthe equation below to estimate KD:

F=p[(KD+LT+n(M))−{(KD+LT+n(M))2−4n(M)(LT)}½]/2+B

where F (mean fluorescence intensity), LT (total mAb binding siteconcentration), p (proportionality constant that relates arbitraryfluorescence units to bound mAb), M (cellular concentration in molarity;0.553 fM based on 100,000 cells in 300 μl), n (number of receptors percell), B (background signal), and KD=equilibrium dissociation constant.

For each antibody titration curve, an estimate for KD was obtained as P,n, B, and KD were floated freely in the nonlinear analysis. For adetailed derivation of the above equation, see Drake and Klakamp (2007),“A rigorous multiple independent binding site model for determiningcell-based equilibrium dissociation constants,” J. Immunol. Methods 318:157-62, which is incorporated by reference herein. Table 3 lists theresulting KD's for all antibodies in order of decreasing affinity alongwith the 95% confidence interval of each fit in parentheses. Theantibody binding site concentration (2× the molecular concentration) wasused for the nonlinear curve-fitting.

Example 10: Affinity Determination by Biacore®

The affinity of IgG antibodies to soluble CD38 ectodomain (ECD) wasdetermined by surface plasmon resonance (SPR) analysis on a Biacore™A100 at 22° C. Goat anti-human IgG polyclonal antibody (Caltag H10500)was immobilized to a CM5 biosensor chip using standard amine coupling tospots 1, 2, 4, and 5 within all four flow cells of the chip.Immobilization levels on each spot ranged from 5865 RU to 6899 RU. HumanCD38 was obtained from R&D Systems (Cat #2404-AC, Lot # PEH020812A). Thestock concentration for CD38 was determined using methods detailed inPace et al. (1995) “How to measure and predict molar absorptioncoefficient of a protein” Protein Science 4(11):2411-23 and Pace andGrimsley (2004) “Spectrophotometric determination of proteinconcentration,” in Current Protocols in Protein Science, Chapter 3: Unit3.1, the teaching of each reference being incorporated by referenceherein.

Running buffer was prepared by degassing HEPES-buffered saline, 0.005%polysorbate 20, and adding filtered BSA to a final concentration of 100μg/mL. All eight purified mAbs were diluted to approximately 2 μg/mLwith running buffer. Preliminary experiments estimated the amount ofeach mAb to be captured in order to maintain a surface capacity(R_(max)) no greater than ˜100 RU. For each mAb capture/antigeninjection cycle, a mAb was captured over spots 1 and 5 within each flowcell with juxtaposed spots 2 and 4 serving as the respective referencesurfaces. Each diluted mAb was captured for 1 minute at a flow rate of10 μL/min followed by three minutes of flowing running buffer forsurface stabilization. HuCD38 was injected over all four flow cells for120 seconds at 30 μL/min over a concentration range of 193.7 nM-3.0 nM(2× serial dilution) followed by a 15 minute dissociation phase. Thesamples were all prepared in the running buffer and were randomlyinjected in triplicate with seven buffer injections interspersed fordouble referencing. The surfaces were regenerated with two 20 secondpulses of 10 mM glycine, pH 1.7.

All sensorgram data were processed with Scrubber 2.0c software andglobally fit to a 1:1 interaction model in Scrubber 2.0c. The resultingbinding constants are shown in Table 2.

TABLE 2 FACS KD FACS KD Biacore Biacore Biacore Anti- (nM) (pM) Ka Kd KDbody MOLP-8 RPMI-8226 (M−1s−1) (s−1) (nM) BM-1 1.1 (0.9) 802  4.49 × 104 2.46 × 10−3 54.8 BM-2 1.6 (0.6) 428  4.24 × 105  2.27 × 10−3 5.4 Ab190.4 (0.3) — 1.54 × 10⁵ 8.10 × 10⁻⁴ 5.3 Ab79 1.2 (1.1) 508 1.22 × 10⁵6.75 × 10⁻⁴ 5.5 Ab72 0.6 (0.4) — 1.44 × 10⁴ 1.82 × 10⁻³ 126 Ab110 1.0(0.1) — 1.22 × 10⁵ 1.71 × 10⁻¹ 1400 Ab43 1.1 (0.3) — 2.72 × 10⁵ 1.46 ×10⁻¹ 537 Ab164 1.4 (0.7) — 1.99 × 10⁵ 7.15 × 10⁻² 359

Example 11: Immunofluorescence Internalization Assays

Immunofluorescence techniques were used to evaluate the internalizationof anti-CD38 antibodies into MOLP-8 cells. MOLP-8 cells were collectedand 5×10⁶ cells were stained for 10 min at 4° C. in RPMI-1640 with 1 μgof each anti-CD38 antibody directly conjugated to Alexa Fluor® 488. Thecells were washed in PBS containing 1% BSA, and 1×10⁶ cells wereincubated for 3 or 6 hours at 4° C. or 37° C. Surface staining wasquenched for 30 min at 4° C. using 2 μg of rabbit anti-Alexa Fluor®-488antibody (Invitrogen). The cells were washed and fixed in PBS with 1%PFA, transferred to a Microtest 96-well plate (BD Biosciences), andeither evaluated by flow cytometry using a FACSCanto™ II (BDBiosciences) flow cytometer or imaged using an ImageXpress® Micro(Molecular Devices) at 20× magnification.

Example 12: Epitope Binning by Biacore®

Biacore® A100 instrumentation was used to bi˜ the two benchmarkantibodies as well as Ab19 and Ab79. The antibodies were firstimmobilized at high and low densities on a CM5 chip using NHS/EDCcoupling chemistry. For each cycle of the epitope binning experiment,CD38 was first injected over these surfaces. Analogous to a sandwichassay in an ELISA format, a unique antibody (taken from the set ofimmobilized antibodies) was then injected over surfaces containingCD38/antibody complexes. Surfaces were regenerated using pulses ofphosphoric acid at the end of each cycle. Data was collected at 22° C.using HBS-P (10 mM 10 HEPES pH 7.4, 150 mM NaCl, 0.005%) P-20)supplemented with BSA. The resulting sensorgrams were processed usingthe “Epitope Mapping” module in the Biacore® A100 Evaluation softwarepackage as well as a trial version of Scrubber for A100 data sets.Replicate data was used to generate a binary 4×4 matrix for the above 4mAbs from two separate experiments, as shown in Table 3.

TABLE 3 Ab79 BM1 Ab19 BM2 Ab79 0 0 1 1 BM1 0 0 1 0 Ab19 1 1 0 0 BM2 1 00 0

Example 13: In Vivo Analysis

The in vivo efficacy of Ab19 and Ab79 was tested in a disseminatedDaudi-luciferase model of human lymphoma. 6-8 week old female CB.17 SCIDmice from Taconic Laboratories were injected intravenously with1×10⁶Daudi-Luc tumor cells. At study day 7, mice were treatedintraperitoneally with: palivizumab, Ab79, Ab19, Benchmark 1, andBenchmark 2. Bioluminescent imaging was performed weekly starting fromday 21 using an IVIS Xenogen system (Caliper Life Sciences) to monitortumor burden. For imaging, animals were injected IP with luciferasesubstrate (150 mg/kg) 10 min before the imaging, then the animals wereanaesthetized under isoflurane and imaged. Results are shown in FIGS. 8and 9.

SEQUENCE LISTING SEQ ID NO: 1 (CD38 Homo sapiens; NP_001766.2)MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEISEQ ID NO: 2 (CD38 Macaca fascicularis; AAT36330.1)MANCEFSPVSGDKPCCRLSRRAQVCLGVCLLVLLILVVVVAVVLPRWRQQWSGSGTTSRFPETVLARCVKYTEVHPEMRHVDCQSVWDAFKGAFISKYPCNITEEDYQPLVKLGTQTVPCNKTLLWSRIKDLAHQFTQVQRDMFTLEDMLLGYLADDLTWCGEFNTFEINYQSCPDWRKDCSNNPVSVFWKTVSRRFAETACGVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQALEAWVIHGGREDSRDLCQDPTIKELESIISKRNIRFFCKNIYRPDKFLQCVKNPEDSSCLSGI SEQ ID NO: 3 (HCDR1 Ab79)GFTFDDYG SEQ ID NO: 4 (HCDR2 Ab79) ISWNGGKT SEQ ID NO: 5 (HCDR3 Ab79)ARGSLFHDSSGFYFGH SEQ ID NO: 6 (LCDR1 Ab79) SSNIGDNYSEQ ID NO: 7 (LCDR2 Ab79) RDS SEQ ID NO: 8 (LCDR3 Ab79) QSYDSSLSGSSEQ ID NO: 9 (Heavy Chain Ab79)EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSDISWNGGKTHYVDSVKGQFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSLFHDSSGFYFGHWGQGTLVTVSSASTKGPSVFPLA SEQ ID NO: 10 (Light Chain Ab79)QSVLTQPPSASGTPGQRVTISCSGSSSNIGDNYVSWYQQLPGTAPKLLIYRDSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDSSLSGSVFGGGTKLTVLGQPKANPTVTLFPPSSEEL SEQ ID NO: 11 (Heavy Chain Ab19)EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYDMTWVRQAPGKGLEWVAVISYDGSDKDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVYYYGFSGPSMDVWGQGTLVTVSSASTKGPSVFPLA SEQ ID NO: 12 (Light Chain Ab19)QSVLTQPPSASGTPGQRVTISCSGSNSNIGSNTVNWYQQLPGTAPKLLIYSDSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDSSLSGSRVFGGGTKLTVLGQPKANPTVTLFPPSSEEL SEQ ID NO: 13 (HCDR1 Ab19) GFTFNNYDSEQ ID NO: 14 (HCDR2 Ab19) ISYDGSDK SEQ ID NO: 15 (HCDR3 Ab19)ARVYYYGFSGPSMDV SEQ ID NO: 16 (LCDR1 Ab19) NSNIGSNTSEQ ID NO: 17 (LCDR2 Ab19) SDS SEQ ID NO: 18 (LCDR3 Ab79) QSYDSSLSGSRSEQ ID NO: 19 (Heavy Chain Ab19) w/constantEVQLLESGGGLVQPGGSLRLSCAASGFTFNNYDMTWVRQAPGKGLEWVAVISYDGSDKDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVYYYGFSGPSMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 20 (Light Chain Ab19) w/constantQSVLTQPPSASGTPGQRVTISCSGSNSNIGSNTVNWYQQLPGTAPKWYSDSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDSSLSGSRVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECSSEQ ID NO: 21 (Heavy Chain Ab79)EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSDISWNGGKTHYVDSVKGQFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSLFHDSSGFYFGHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 22 (Light Chain Ab79)QSVLTQPPSASGTPGQRVTISCSGSSSNIGDNYVSWYQQLPGTAPKLLIYRDSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDSSLSGSVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP TECSSEQ ID NO: 23 (CD157 Homo sapiens; NP_004325)MAAQGCAASRLLQLLLQLLLLLLLLAAGGARARWRGEGTSAHLRDIFLGRCAEYRALLSPEQRNKNCTAIWEAFKVALDKDPCSVLPSDYDLFINLSRHSIPRDKSLFWENSHLLVNSFADNTRRFMPLSDVLYGRVADFLSWCRQKNDSGLDYQ SCPTSEDCENNPVDSFWKRASIQYSKDSSGVIHVMLNGSEPTGAYPIKGFFADYEIPNLQKEKITRIEIWVMHEIGGPNVESCGEGSMKVLEKRLKDMGFQYSCINDYRPVKLLQCVDHSTHPDCALKSAAAATQRKAPSLYTEQRAGLIIPLFLVLASRTQLSEQ ID NO: 24 (Benchmark 1; Heavy Chain Variable Region)EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWF GEPVFDYWGQGTLVTVSSSEQ ID NO: 25 (Benchmark 1; Light Chain Variable Region)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK RSEQ ID NO: 26 (Benchmark 2; Heavy Chain Variable Region)QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYWMQWVKQRPGQGLEWIGTIYPGDGDTGYAQKFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGDY YGSNSLDYWGQGTSVTVSSSEQ ID NO: 27 (Benchmark 2; Light Chain Variable Region)DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQSPRRLIYSASYRYIGVPDRFTGSGAGTDFTFTIS SVQAEDLAVYYCQQHYSPPYTFGGGTKLEI KRSEQ ID NO: 28 (Heavy Chain Ab43)EVQLLESGGGLVQPGGSLRLSCAASGFTESSYGMHWVRQAPGKGLEWVSRINSDGSSTSYADSMKGQFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGYYYYAMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 29 (Light Chain Ab43)QSVLTQPPSASGTPGQRVTISCSGGSSNIGYKTVNWYQQLPGTAPKLLIYDNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGLVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECSSEQ ID NO: 30 (Heavy Chain Ab72)EVQLLESGGGLVQPGGSLRLSCAASGFTESSYGMNWVRQAPGKGLEWVSGISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSNYDFWSGYYYGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 31 (Light Chain Ab72)QSVLTQPPSASGTPGQRVTISCSGSSSNIGSKTVSWYQQLPGTAPKLLIYDNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYAARSTNIIFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP TECSSEQ ID NO: 32 (Heavy Chain Ab110)EVQLLESGGGLVQPGGSLRLSCAASGFTESSYGMHWVRQAPGKGLEWVSITYSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRATWGGATHDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 33 (Light Chain Ab110)QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDDSLNGVLFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECSSEQ ID NO: 34 (Heavy Chain Ab19) w/constantEVQLLESGGGLVQPGGSLRLSCAASGFTFNNYDMTWVRQAPGKGLEWVAVISYDGSDKDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVYYYGFSGPSMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 35 (Light Chain Ab19) w/constantQSVLTQPPSASGTPGQRVTISCSGSNSNIGSNTVNWYQQLPGTAPKLLIYSDSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQSYDSSLSGSRVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS

Having described the invention in detail and by reference to specificembodiments thereof, it will be apparent that modifications andvariations are possible without departing from the scope of theinvention defined in the appended claims. More specifically, althoughsome aspects of the present invention are identified herein asparticularly advantageous, it is contemplated that the present inventionis not necessarily limited to these particular aspects of the invention.

1.-21. (canceled)
 22. An isolated antibody which specifically bindshuman CD38 (SEQ ID NO:1) and cynomolgus CD38 (SEQ ID NO:2), wherein theheavy chain variable region of the antibody comprises an amino acidsequence having an identity of at least 90% to SEQ ID NO:9, and thelight chain variable region of the antibody comprises an amino acidsequence having an identity of at least 90% to SEQ ID NO:10; wherein theantibody binds to human CD38 (SEQ ID NO:1) with a KD of 10⁻⁸ M or agreater affinity, and wherein the affinity is measured by a standardBiacore assay; and wherein the antibody is covalently attached to a drugmoiety.
 23. The isolated antibody of claim 22, wherein the heavy chainvariable region comprises an amino acid sequence having an identity ofat least 95% to SEQ ID NO:9, and the light chain variable regioncomprises an amino acid sequence having an identity of at least 95% toSEQ ID NO:10.
 24. The isolated antibody of claim 22, further comprisingan Fc domain.
 25. The isolated antibody of claim 24, wherein the Fcdomain is a human Fc domain.
 26. The isolated antibody of claim 25,wherein the Fc domain is a variant Fc domain.
 27. The isolated antibodyof claim 22, wherein the drug moiety is attached to the antibody using alinker.
 28. The isolated antibody of claim 27, wherein the drug moietyis selected from the group consisting of an auristatin, a maytansinoid,a calicheamicin, a dolastatin, and a trichothecene.
 29. An isolatedantibody which specifically binds human CD38 (SEQ ID NO:1) andcynomolgus CD38 (SEQ ID NO:2), wherein the heavy chain variable regionof the antibody comprises an amino acid sequence having an identity ofat least 90% to SEQ ID NO:11, and the light chain variable region of theantibody comprises an amino acid sequence having an identity of at least90% to SEQ ID NO:12; wherein the antibody binds to human CD38 (SEQ IDNO:1) with a KD of 10⁻⁸ M or a greater affinity, and wherein theaffinity is measured by a standard Biacore assay; and wherein theantibody is covalently attached to a drug moiety.
 30. The isolatedantibody of claim 29, wherein the heavy chain variable region comprisesan amino acid sequence having an identity of at least 95% to SEQ IDNO:11, and the light chain variable region comprises an amino acidsequence having an identity of at least 95% to SEQ ID NO:12.
 31. Theisolated antibody of claim 29, further comprising an Fc domain.
 32. Theisolated antibody of claim 31, wherein the Fc domain is a human Fcdomain.
 33. The isolated antibody of claim 32, wherein the Fc domain isa variant Fc domain.
 34. The isolated antibody of claim 29, wherein thedrug moiety is attached to the antibody using a linker.
 35. The isolatedantibody of claim 34, wherein the drug moiety is selected from the groupconsisting of an auristatin, a maytansinoid, a calicheamicin, adolastatin, and a trichothecene.
 36. A method of treating cancer cellsexpressing CD38 comprising administering to a patient in need thereof anisolated antibody that specifically binds human CD38 (SEQ ID NO:1) andcynomolgus CD38 (SEQ ID NO:2), comprising: a) a heavy chain variableregion comprising an amino acid sequence having an identity of at least90% to SEQ ID NO:9, b) a light chain variable region comprising an aminoacid sequence having an identity of at least 90% to SEQ ID NO:10; and c)a covalently attached drug moiety, wherein the antibody binds to humanCD38 (SEQ ID NO:1) with a KD of 10⁻⁸ M or a greater affinity, andwherein the affinity is measured by a standard Biacore assay.
 37. Themethod of claim 36, wherein the heavy chain variable region comprises anamino acid sequence having an identity of at least 95% to SEQ ID NO:9,and the light chain variable region comprises an amino acid sequencehaving an identity of at least 95% to SEQ ID NO:10.
 38. An isolatednucleic acid encoding the heavy chain variable region of claim
 22. 39.An isolated nucleic acid encoding the light chain variable region ofclaim
 22. 40. An isolated nucleic acid composition comprising: a) afirst nucleic acid encoding the heavy chain variable region of claim 22;and b) a second nucleic acid encoding the light chain variable region ofclaim
 22. 41. An expression vector comprising the nucleic acid of claim38 and the nucleic acid of claim
 39. 42. A host cell comprising theisolated nucleic acid composition of claim
 40. 43. A host cellcomprising the expression vector of claim
 41. 44. A method of producingan antibody that specifically binds human CD38 (SEQ ID NO:1) andcynomolgus CD38 (SEQ ID NO:2), comprising culturing the host cell ofclaim 42, wherein the antibody is produced.
 45. A method of producing anantibody that specifically binds human CD38 (SEQ ID NO:1) and cynomolgusCD38 (SEQ ID NO:2), comprising culturing the host cell of claim 43,wherein the antibody is produced.
 46. An isolated nucleic acid encodingthe heavy chain variable region of claim
 29. 47. An isolated nucleicacid encoding the light chain variable region of claim
 29. 48. Anisolated nucleic acid composition comprising: a) a first nucleic acidencoding the heavy chain variable region of claim 29; and b) a secondnucleic acid encoding the light chain variable region of claim
 29. 49.An expression vector comprising the nucleic acid of claim 46 and thenucleic acid of claim
 47. 50. A host cell comprising the isolatednucleic acid composition of claim
 48. 51. A host cell comprising theexpression vector of claim
 49. 52. A method of producing an antibodythat specifically binds human CD38 (SEQ ID NO:1) and cynomolgus CD38(SEQ ID NO:2), comprising culturing the host cell of claim 50, whereinthe antibody is produced.
 53. A method of producing an antibody thatspecifically binds human CD38 (SEQ ID NO:1) and cynomolgus CD38 (SEQ IDNO:2), comprising culturing the host cell of claim 51, wherein theantibody is produced.